Enzymatic peracid production using a cosolvent

ABSTRACT

Disclosed herein are two-component enzymatic peracid generation systems and methods of using such systems wherein the first component comprises a formulation of at least one enzyme catalyst having perhydrolysis activity, a carboxylic acid ester substrate, and a cosolvent and wherein the second component comprises a source of peroxygen in water. The two components are combined to produce an aqueous peracid formulation useful as, e.g., a disinfecting or bleaching agent. Specifically, organic cosolvents are used to control the viscosity of a substrate-containing component and to enhance the solubility of the substrate in an aqueous reaction formulation without causing substantial loss of perhydrolytic activity of the enzyme catalyst.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/102,505; 61/102,512; 61/102,514; 61/102,520; 61/102,531; and61/102,539; each filed Oct. 3, 2008, each of which incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The following relates to the field of enzymatic peracid synthesis and insitu enzyme catalysis using multicomponent systems. Specifically,processes are provided to produce and efficaciously deliverperoxycarboxylic acids using the perhydrolysis activity of enzymesidentified structurally as belonging to the CE-7 family of carbohydrateesterases, including cephalosporin acetyl hydrolases (CAHs; E.C.3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72) usingmulti-component systems (i.e., in the present context, systems thatinvolve the production of peroxycarboxylic acid using at least tworeaction components that are separately stored prior to a desired timeof reaction). At least one peroxycarboxylic acid is produced atsufficient concentrations as to be efficacious for the disinfection orsanitization of surfaces, medical instrument sterilization, foodprocessing equipment sterilization, and suitable for use in laundry careapplications such as disinfecting, bleaching, destaining, deodorizing,and sanitizing.

BACKGROUND OF THE INVENTION

Peracid compositions have been reported to be effective antimicrobialagents. Methods to clean, disinfect, and/or sanitize hard surfaces, meatproducts, living plant tissues, and medical devices against undesirablemicrobial growth have been described (e.g., U.S. Pat. No. 6,545,047;U.S. Pat. No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. Pat. No.5,683,724; and U.S. Patent Application Publication No, 2003/0026846).Peracids have also been reported to be useful in preparing bleachingcompositions for laundry detergent applications (U.S. Pat. No.3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No. 5,364,554).

Peroxycarboxylic acids can be prepared by the chemical reaction of acarboxylic acid alkyl ester and a peroxide reagent, such as hydrogenperoxide (see Organic Peroxides, Daniel Swern, ed., Vol. 1, pp 313-516;Wiley Interscience, New York, 1971), However, under slightly basic toacidic pH (from about 8 to about 4), the reaction often does not proceedrapidly enough to produce a peroxycarboxylic acid concentration that issuitable for many commercial disinfecting and/or bleaching applications.

One way to overcome the disadvantages of chemical peroxycarboxylic acidproduction is to use an enzyme catalyst having perhydrolysis activity.U.S. patent application Ser. No. 11/638,635 and U.S. Patent ApplicationPublication Nos. 2008/0176783; 2008/0176299; and 2009/0005590 toDiCosimo et al. disclose enzymes structurally classified as members ofthe CE-7 family of carbohydrate esterases (e.g., cephalosporin Cdeacetylases [CAHs] and acetyl xylan esterases [AXEs]) that arecharacterized by significant perhydrolysis activity for convertingcarboxylic acid esters (in the presence of a suitable source ofperoxygen, such as hydrogen peroxide) into peroxycarboxylic acids atconcentrations sufficient for use as a disinfectant and/or a bleachingagent. Some members of the CE-7 family of carbohydrate esterases havebeen demonstrated to have perhydrolytic activity sufficient to produce4000-5000 ppm peracetic acid from acetyl esters of alcohols, diols, andglycerols in 1 minute and up to 9000 ppm between 5 minutes and 30minutes once the reaction components were mixed (DiCosimo of al., U.S.2009/0005590). The enzymatic peracid generation system described byDiCosimo et al. in each of the cited patent application publications maybe based on the use of multiple reaction components that remainseparated until the peracid solution is needed.

It has been observed that, when using a multi-component systemcomprising a first enzyme catalyst/substrate component and a secondcomponent comprising an aqueous source of peroxygen, the use of one ormore substrates that are insoluble or partially insoluble in water aftermixing of the two components can result in at least three conditionsthat interfere with the ability to efficaciously produce and deliver aperoxycarboxylic acid product: first, the viscosity of the enzymecatalyst/substrate constituent can be too high to permit efficientmixing with a second constituent comprising a source of peroxygen, whichdecreases the rate of production of peroxycarboxylic acid; second, theviscosity of the enzyme catalyst/substrate constituent can be too highto permit certain modes of delivery of a product comprising a mixture ofthe enzyme catalyst/substrate constituent and the source of peroxygen,such as spraying; third, the dissolution rate of the substrate in theenzyme/substrate component after mixing with a second componentcomprising a source of peroxygen in aqueous solution is too low topermit a satisfactory rate of production of peroxycarboxylic acid. Theseproblems also become evident in situations where use of a particularratio of a component comprising an aqueous source of peroxygen to acomponent comprising an enzyme catalyst/substrate constituent isdesired. As such, commercial uses of multi-component systems thatinvolve the storage of the enzyme catalyst having perhydrolysis activityand substrate separately from a source of peroxygen until a desired timeof reaction have remained impracticable for some applications.

The problem to be solved is to provide a method to enzymatically produceperacids when using a multi-component generation system characterized byat least one first component comprising a formulation of a carboxylicacid ester substrate and an enzyme catalyst comprising a CE-7carbohydrate esterase having perhydrolysis activity, wherein thecarboxylic acid ester substrate is (1) partially or substantiallyinsoluble in an aqueous matrix, and/or (2) slow to dissolve into anaqueous reaction matrix and/or (3) has a viscosity that does notfacilitate easy mixing for some commercial applications (e.g., use of atwo compartment spray bottle designed to mix two liquid componentshaving different viscosities and/or solubilities), and additionallycharacterized by at least one second component comprising an aqueoussolution comprising a source of peroxygen (e.g., an aqueous formulationof hydrogen peroxide).

SUMMARY OF THE INVENTION

The problem has been solved by providing a method and a system toenzymatically produce a peracid that incorporates the use of at leastone cosolvent in the first component of a two component system, whereinsaid first component is a substantially non-aqueous formulation of acarboxylic acid ester and at least one enzyme catalyst comprising a CE-7carbohydrate esterase having perhydrolysis activity, wherein theaddition of the at least one cosolvent improves the viscosity of thefirst component for delivery and mixing in a two component system,allows for the desired adjustment of the volume of the first componentso as to enable mixing with the second component with a desired ratio ofthe two components, and improves the solubility and/or dissolution rateof the first component when combined with an aqueous second component(i.e., an aqueous formulation providing a source of peroxygen) to forman aqueous peracid formulation.

It has been discovered that the inclusion of a cosolvent comprising anorganic solvent having a log P of less than about 2, wherein log P isdefined as the logarithm of the partition coefficient of a substancebetween octanol and water, expressed as log P=log[[solute]_(octanol)/[solute]_(water)], resolves the aforementionedconditions that otherwise interfere with the ability to efficaciouslyproduce and deliver a peroxycarboxylic acid product in a form that canbe delivered by means that are conventional to or otherwise suitable inconsumer, industrial, and medical contexts. The cosolvent is preferablyinert and nonreactive in the formulation and miscible with thecarboxylic acid ester substrate, wherein the cosolvent is not asubstrate for said enzyme catalyst. The cosolvent is preferably anorganic alcohol lacking an enzymatically perhydrolyzable ester group. Asused herein, an “alcohol” is molecule comprising at least one hydroxylmoiety. The cosolvent also is preferably soluble at its finalconcentration following mixing or contacting the components of themulti-component system. However, it has been reported that biocatalysisin organic solvents having the having a log P of less than about 2 oftenadversely affects enzyme activity (for example, by inactivating theenzyme constituent (see, e.g., C. Laane at, Biotechnol. Bioeng. 30:81-87(1987) and Cowan, D. A. and Plant, A., Biocatalysis in Organic PhaseSystems., Ch 7 in Biocatalysis at Extreme Temperatures, Kelly, R. W. W.and Adams, M., eds., Amer. Chem. Soc. Symposium Series, OxfordUniversity Press, New York, N.Y., pp 86-107 (1992)). As such, thebeneficial results provided by the inclusion of a cosolvent comprisingan alcohol represent an unexpectedly positive outcome. These and otherbenefits of the present methods and systems are discussed more fullyinfra.

In one embodiment, a method for producing a peroxycarboxylic acid isprovided comprising

(a) providing a first component comprising:

-   -   (i) a carboxylic acid ester substrate;    -   (ii) an enzyme catalyst having perhydrolysis activity, wherein        said enzyme catalyst comprises an enzyme having a carbohydrate        esterase family 7 (CE-7) signature motif that aligns with SEQ ID        NO: 2 using CLUSTALW, said signature motif comprising;        -   (1) an RGQ motif at amino acid positions aligning with            118-120 of SEQ ID NO:2;        -   (2) a GXSQG motif at amino acid positions aligning with            179-183 of SEQ ID NO:2; and        -   (3) an HE motif at amino acid positions aligning with            298-299 of SEQ ID NO:2;    -   said enzyme comprising at least 30% amino acid identity to SEQ        ID NO: 2; and    -   (iii) at least one cosolvent comprising an organic solvent        having a log P of less than about 2, wherein log P is defined as        the logarithm of the partition coefficient of a substance        between octanol and water, expressed as P        [solute]_(octanol)/[solute]_(water) and wherein the at least one        cosolvent is not a substrate for said enzyme catalyst;

wherein said first component is a substantially non-aqueous mixture of(i)-(iii);

(b) providing a second component comprising a source of peroxygen inwater; and

(c) combining said first component and said second component to form anaqueous reaction mixture,

wherein said cosolvent solubilizes the carboxylic acid ester substratein the aqueous reaction mixture without substantial loss ofperhydrolytic activity of the enzyme catalyst and wherebyperoxycarboxylic acid is produced.

In another aspect, a method for disinfecting a surface comprisingperforming the method above, further comprising the step of applyingsaid aqueous reaction mixture comprising peroxycarboxylic acid to asurface for disinfection or bleaching.

In another aspect, a method for treating an article of clothing isprovided comprising performing the method above, further comprising thestep of applying said aqueous reaction mixture comprisingperoxycarboxylic acid to the article of clothing for bleaching, stainremoval, odor reduction, sanitization, disinfection, or a combinationthereof.

Another aspect of the invention is a multi-component system forproducing a peroxycarboxylic acid comprising

(a) providing a first component comprising:

-   -   (i) a carboxylic acid ester substrate;    -   (ii) an enzyme catalyst having perhydrolysis activity, wherein        said enzyme catalyst comprises an enzyme having a CE-7 signature        motif that aligns with SEQ ID NO: 2 using CLUSTALW, said        signature motif comprising;        -   (1) an RGQ motif at amino acid positions aligning with            118-120 of SEQ ID NO: 2;        -   (2) a GXSQG motif at amino acid positions aligning with            179-183 of SEQ ID NO: 2; and        -   (3) an HE motif at amino acid positions aligning with            298-299 of SEQ ID NO: 2;    -   said enzyme comprising at least 30% amino acid identity to SEQ        ID NO: 2; and    -   (iii) at least one cosolvent comprising an organic solvent        having a log P of less than about 2, wherein log P is defined as        the logarithm of the partition coefficient of a substance        between octanol and water, expressed as        P=[solute]_(octanol)/[solute]_(water) and wherein the cosolvent        is not a substrate for said enzyme catalyst;

wherein said first component is a substantially non-aqueous mixture of(i)-(iii); and

(b) providing a second component comprising a source of peroxygen inwater;

wherein said first component and said second component are combined toproduce an aqueous reaction mixture comprising a peroxycarboxylic acidand wherein said cosolvent solubilizes the carboxylic acid estersubstrate in the aqueous reaction mixture without substantial loss ofperhydrolytic activity of the enzyme catalyst.

The design of the multi-component packaging/delivery systems forseparating and combining the first and second components describedherein will generally depend upon the physical form of the individualreaction components. In one embodiment, any delivery system suitable forliquid-liquid systems may be used. In another embodiment, the presenttwo-component peracid generation system comprises a first containercomprising the present first component and a second container comprisingthe present second component. Typical examples of containers and/orpacking system may include individual bottles, individual bottlespackaged together in a single kit, dual chamber dispenser bottles (suchas squeeze bottles, spray bottles, and the like.), rigid or non-rigiddual chamber dispenser packets, individual packets, and dissolvable ordegradable dual chamber dispenser packets, to name a few. (See U.S. Pat.Nos. 4,678,103; 4,585,150; 6,223,942; 5,954,213; 6,758,411; 5,862,949;5,398,846; 6,995,125; and 6,391,840; U.S. Published Patent ApplicationNos. 2005/0139608 and 2002/0030063; and PCT Publication Nos. WO00/61713;WO02/22467; and WO2005/035705). In one embodiment, the delivery systemis a dual compartment spray bottle.

In further embodiments, the enzyme catalyst comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO: 26, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 64, SEQID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74 or asubstantially similar enzyme having perhydrolase activity derived bysubstituting, deleting or adding one or more amino acids to said aminoacid sequence.

In a further embodiment, the substantially similar enzyme havingperhydrolase activity is at least 30%, preferably at least 33%, morepreferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, yet even more preferable at least 70%, yet evenmore preferably at least 80%, yet even more preferably at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or moreamino acid sequences selected from the group consisting of SEQ ID NO: 2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQID NO:24, SEQ ID NO: 26, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 64,SEQ ID NO; 68, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74.

In one aspect, the carboxylic acid ester substrate used in the presentmethods and multi-component systems is selected from the groupconsisting of:

-   -   (a) one or more esters having the structure        [X]_(m)R₅    -   wherein        -   X is an ester group of the formula R₆C(O)O;    -   R₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with a hydroxyl group or C1 to C4 alkoxy        group, wherein R₆ optionally comprises one or more ether        linkages where R₆ is C2 to C7;        -   R₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl            moiety optionally substituted with a hydroxyl group, wherein            each carbon atom in R₅ individually comprises no more than            one hydroxyl group or no more than one ester group, and            wherein R₅ optionally comprises one or more ether linkages;    -   m is 1 to the number of carbon atoms in R₅,    -   said one or more esters having solubility in water of at least 5        ppm at 25° C.;    -   (b) one or more glycerides having the structure

-   -   -   wherein R₁ is a C1 to C7 straight chain or branched chain            alkyl optionally substituted with a hydroxyl or a C1 to C4            alkoxy group and R₃ and R₄ are individually H or R₁C(O);

    -   (c) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10;    -   (d) one or more acetylated monosaccharides, acetylated        disaccharides, or acetylated polysaccharides; and    -   (e) any combination of (a) through (d);

In one embodiment, the carboxylic acid ester substrate is selected fromthe group consisting of; monoacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylanfragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-glucal; propylene glycoldiacetate, ethylene glycol diacetate; monoesters or diesters of1,2-ethanediol; 1,2-propanediol; 1,3-propanediol; 1,2-butanediol;1,3-butanediol; 2,3-butanediol; 1,4-butanediol; 1,2-pentanediol;2,5-pentanediol; 1,6-pentanediol; 1,2-hexanediol; 2,5-hexanediol;1,6-hexanediol; and mixtures thereof. In another embodiment thesubstrate is selected from the group consisting of propylene glycoldiacetate, ethylene glycol diacetate, and mixtures thereof. In furtherembodiment, the substrate preferably comprises triacetin.

Unless otherwise specified, disclosure of a particular embodimentapplies equally to the present methods for producing peroxycarboxylicacid in a multi-component system, the present methods for disinfecting asurface, the present multi-component formulation compositions, and thepresent generation systems for producing peroxycarboxylic acid.

In a further aspect, the present method and systems may also be used inlaundry care applications to produce a beneficial effect including, butnot limited to, bleaching, destaining, deodorizing, and sanitizing.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1, sheets A-F, show the results of a CLUSTALW alignment (version1.83) of several enzymes structurally classified as carbohydrateesterase family 7 members including SEQ ID NOs 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 54, 60, 64, 68, 72, 73, and 74. All of theenzymes share the conserved motifs (underlined) that together form thesignature motif for CE-7 carbohydrate esterases (see Vincent of al., J.Mol. Biol., 330:593-606 (2003) and U.S. Patent Application PublicationNo. 2008/0176783 to DiCosimo et al.). An additional motif that may beused to further characterize members of the CE-7 family of enzymes isunderlined and bold (i.e., the LXD motif).

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleic acid sequence of the cephalosporin Cdeacetylase (cah) coding region from Bacillus subtilis ATCC® 31954™.

SEQ ID NO: 2 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus subtilis ATCC® 31954™.

SEQ ID NO: 3 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from B. subtilis subsp. subtilis str. 168.

SEQ ID NO: 4 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from B. subtilis subsp. subtilis str. 168, and is identicalto the deduced amino acid sequence of the cephalosporin C deacetylasefrom B. subtilis BE1010.

SEQ ID NO: 5 is the nucleic acid sequence of the cephalosporinacetylesterase coding region from B. subtilis ATCC® 6633™.

SEQ ID NO: 6 is the deduced amino acid sequence of the cephalosporinacetylesterase from B. subtilis ATCC® 6633™.

SEQ ID NO: 7 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from B. licheniformis ATCC® 14580™.

SEQ ID NO: 8 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from B. licheniformis ATCC® 14580™.

SEQ ID NO: 9 is the nucleic acid sequence of the acetyl xylan esterasecoding region from B. pumilus PS213.

SEQ ID NO: 10 is the deduced amino acid sequence of the acetyl xylanesterase from B. pumilus PS213.

SEQ ID NO: 11 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Clostridium thermocellum ATCC® 27405™.

SEQ ID NO: 12 is the deduced amino acid sequence of the acetyl xylanesterase from Clostridium thermocellum ATCC® 27405™.

SEQ ID NO: 13 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga neapolitana.

SEQ ID NO: 14 is the deduced amino acid sequence of the acetyl xylanesterase from Thermotoga neapolitana.

SEQ ID NO: 15 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga maritima MSB8.

SEQ ID NO: 16 is the deduced amino acid sequence of the acetyl xylanesterase from Thermotoga maritima MSB8.

SEQ ID NO: 17 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 18 is the deduced amino acid sequence of the acetyl xylanesterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 19 is the nucleic acid sequence encoding the cephalosporin Cdeacetylase from Bacillus sp. NRRL B-14911 as reported in GENBANK®Accession number ZP_(—)01168674. However, the reported sequence appearsto have a 15 amino acid N-terminal addition that is likely incorrectbased on sequence alignments with other cephalosporin C deacetylases anda comparison of the reported length (340 amino acids) versus theobserved length of other CAH enzymes (typically 318-325 amino acids inlength).

SEQ ID NO: 20 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus sp. NRRL B-14911 without the N-terminal 15amino acids reported under GENBANK® Accession number ZP_(—)01168674.

SEQ ID NO: 21 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from Bacillus halodurans C-125.

SEQ ID NO: 22 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus halodurans C-125.

SEQ ID NO: 23 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from Bacillus clausii KSM-K16.

SEQ ID NO: 24 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 25 is the nucleic acid sequence of the Bacillus subtilis ATCC29233™ cephalosporin C deacetylase (cab) gene cloned into pSW190.

SEQ ID NO: 26 is the deduced amino acid sequence of the Bacillussubtilis ATCC® 29233™ cephalosporin C deacetylase (CAH).

SEQ ID NOs: 27 and 28 are primers used to PCR amplify the Thermotoganeapolitana acetyl xylan esterase coding region (GENBANK® U58632) forconstruction of pSW196.

SEQ ID NO: 29 is the nucleic acid sequence of the codon-optimizedversion of the Thermotoga neapolitana acetyl xylan esterase gene inplasmid pSW196.

SEQ ID NO: 30 is the nucleic acid sequence of the kanamycin resistancegene (kan).

SEQ ID NO: 31 is the nucleic acid sequence of plasmid pKD13.

SEQ ID NOs: 32 and 33 are primers used to generate a PCR productencoding the kanamycin gene flanked by regions having homology to thekatG catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katG gene.

SEQ ID NO: 34 is the nucleic acid sequence of the PCR product encodingthe kanamycin resistance gene flanked by regions having homology to thekatG catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katG gene.

SEQ ID NO: 35 is the nucleic acid sequence of the katG catalase gene inE. coli MG1655.

SEQ ID NO: 36 is the deduced amino acid sequence of the KatG catalase inE. coli MG1655.

SEQ ID NO: 37 is the nucleic acid sequence of plasmid pKD46.

SEQ ID NOs: 38 and 39 are primers used to confirm the disruption of thekatG gene.

SEQ ID NO: 40 is the nucleic acid sequence of plasmid pCP20.

SEQ ID NOs: 41 and 42 are primers used to generate a PCR productencoding the kanamycin gene flanked by regions having homology to thekatE catalase gene in E. coli MG1655. The product was; used to disruptthe endogenous katE gene.

SEQ ID NO: 43 is the nucleic acid sequence of the PCR product encodingthe kanamycin resistance gene flanked by regions having homology to thekatE catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katE gene.

SEQ ID NO: 44 is the nucleic acid sequence of the katE catalase gene inE. coli MG1655.

SEQ ID NO: 45 is the deduced amino acid sequence of the KatE catalase inE. coli MG1655.

SEQ ID NOs: 46 and 47 are primers used to confirm disruption of the katEgene in the single knockout strain E. coli MG1655 ΔkatE, and in thedouble-knockout strain E. coli MG1655 ΔkatG ΔkatE, herein referred to asE. coli KLP18.

SEQ ID NO: 48 is the nucleic acid sequence of the codon optimizedversion of the Bacillus pumilus PS213 encoding the amino acid sequenceSEQ ID NO: 10.

SEQ ID NO: 49 is the amino acid sequence of the region encompassingamino acids residues 118 through 299 of SEQ ID NO: 2.

SEQ ID NO: 50 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Bacillus clausii KSM-K16cephalosporin-C deacetylase coding sequence.

SEQ ID NO: 51 is the nucleic acid sequence of the codon-optimizedBacillus clausii KSM-K16 cephalosporin-C deacetylase coding sequence.

SEQ ID NO: 52 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermoanaerobacterium saccharolyticumacetyl xylan esterase coding sequence.

SEQ ID NO: 53 is the nucleic acid sequence of the codon-optimizedversion of the Thermoanaerobacterium saccharolyticum acetyl xylanesterase coding sequence.

SEQ ID NO: 54 is the deduced amino acid sequence of the acetyl xylanesterase from Thermoanaerobacterium saccharolyticum (GENBANK®AccessionNo. S41858).

SEQ ID NO: 55 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga maritima MSB8 acetyl xylanesterase coding sequence.

SEQ ID NO: 56 is the nucleic acid sequence of the codon-optimizedversion of the Thermotoga maritima MSB8 acetyl xylan esterase codingsequence.

SEQ ID NO: 57 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga lettingae acetyl xylanesterase coding sequence.

SEQ ID NO: 58 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga lettingae acetyl xylanesterase coding sequence.

SEQ ID NO: 59 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga lettingae.

SEQ ID NO: 60 is the deduced amino acid sequence of the acetyl xylanesterase from Thermotoga lettingae.

SEQ ID NO: 61 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga petrophila acetyl xylanesterase coding sequence.

SEQ ID NO: 62 is the nucleic acid sequence of the PCR product encoding acodon-optimized version of the Thermotoga petrophila acetyl xylanesterase coding sequence.

SEQ ID NO: 63 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga petrophila.

SEQ ID NO: 64 is the deduced amino acid sequence of an acetyl xylanesterase from Thermotoga petrophila

SEQ ID NO: 65 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga sp. RQ2 “RQ2(a)” acetylxylan esterase coding sequence.

SEQ ID NO: 66 is the nucleic acid sequence of the PCR product encoding acodon-optimized version of the Thermotoga sp. RQ2 “RQ2(a)” acetyl xylanesterase coding sequence.

SEQ ID NO: 67 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga sp. RQ2 identified herein as “RQ2(a)”.

SEQ ID NO: 68 is the deduced amino acid sequence of an acetyl xylanesterase (GENBANK® Accession No. ACB09222) from Thermotoga sp. RQ2identified herein as “RQ2(a)”.

SEQ ID NO: 69 is the nucleic acid sequence of the PCR product encodingthe codon-optimized version of the Thermotoga sp. RQ2 “RQ2(b)” acetylxylan esterase coding sequence.

SEQ ID NO: 70 is the nucleic acid sequence of the PCR product encoding acodon-optimized version of the Thermotoga sp. RQ2 “RQ2(b)” acetyl xylanesterase coding sequence.

SEQ ID NO: 71 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga sp. RQ2 identified herein as “RQ2(b)”.

SEQ ID NO: 72 is the deduced amino acid sequence of an acetyl xylanesterase (GENBANK® Accession No. ACB08860) from Thermotoga sp. RQ2identified herein as “RQ2(b)”.

SEQ ID NO: 73 is the deduced amino acid sequence of a Thermotoganeapolitana acetyl xylan esterase variant from co-owned, co-filed, andcopending U.S. patent application Attorney Docket No. CL4392 US NA(incorporated herein by reference in its entirety), where the Xaaresidue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 74 is the deduced amino acid sequence of a Thermotogamaritima MSB8 acetyl xylan esterase variant from co-owned, co-filed, andcopending U.S. patent application Attorney Docket No. CL4392 US NA,where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

DETAILED DESCRIPTION OF THE INVENTION

In certain applications for a multicomponent in situ peraciddisinfectant formulation, it may be desirable for the ratio of thesecond component (comprising an aqueous source of peroxygen) to thefirst component (comprising the enzyme and the enzyme substrate) to bewithin a range of from 1:1 to 10:1, where from 10 parts to 1 part (byweight) of the second component is mixed with one part (by weight) ofthe first component to produce a peracid at a concentration efficaciousfor disinfection.

The stated problems have been solved by the discovery that, inmulticomponent methods and systems in the present context, methods andsystems that involve the production of peroxycarboxylic acid using atleast two reaction components that are separately stored prior to adesired time of reaction), substrates of perhydrolases can beefficaciously mixed with a source of peroxygen in water andsatisfactorily delivered, for example, to a surface, through theinclusion of a cosolvent. As described herein, addition of an organicsolvent to a catalyst composition (Component A; herein also referred toas the “first” component) and removal of an equal volume of water fromthe aqueous source of peroxygen (Component B; herein also referred to asthe “second” component), such that the ratio of Component B to ComponentA is within a range of from 1:1 to 10:1, allows production of a peracidat a concentration efficacious for disinfection. The organic solvent ispreferably inert and non-reactive in the formulation. The cosolvent alsois preferably completely miscible with the enzyme substrate (e.g.,triacetin). The cosolvent also is preferably soluble at its finalconcentration in the combined Components A and B after mixing. Pursuantto the present invention, the CE-7 carbohydrate esterase family ofstructurally-related enzymes can be used in multicomponent systems togenerate concentrations of peracids with high efficiency fordisinfection and/or bleaching applications, and, as described for fullyinfra, cosolvents comprising organic solvents can surprisingly be usedto enhance the solubility of the substrate in an aqueous reactionformulation without substantial loss of perhydrolytic activity of theenzyme catalyst. In a further aspect, the present invention includesmethods and multi-component systems for use in laundry care applicationswherein an article of clothing or a textile is contacted with peracidsat concentrations suitable for bleaching, stain removal, odor reduction,sanitization, disinfection, or a combination thereof.

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingFIGURE, the sequence listing, and examples, which form a part of thisdisclosure. It is to be understood that this invention is not limited tothe specific products, methods, conditions or parameters describedand/or shown herein, and that the terminology used herein is for thepurpose of describing particular embodiments by way of example only andis not intended to be limiting of the claimed invention.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an” and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of” and “consisting of”. Similarly, the term“consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “peracid” is synonymous with peroxyacid,peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoicacid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the terms “substrate”, “suitable substrate”, and“carboxylic acid ester substrate” interchangeably refer specifically to:

-   -   (a) one or more esters having the structure        [X]_(m)R₅    -   wherein    -   X is an ester group of the formula R₆C(O)O;    -   R₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with a hydroxyl group or C1 to C4 alkoxy        group, wherein R₅ optionally comprises one or more ether        linkages where R₆ is C2 to C7;    -   R₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety        optionally substituted with a hydroxyl group, wherein each        carbon atom in R₅ individually comprises no more than one        hydroxyl group or no more than one ester group, and wherein R₅        optionally comprises one or more ether linkages;    -   m is 1 to the number of carbon atoms in R₅,    -   said one or more esters having solubility in water of at least 5        ppm at 25° C.; or    -   (b) one or more glycerides having the structure

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O); or    -   (c) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; or    -   (d) one or more acetylated monosaccharides, acetylated        disaccharides, or acetylated polysaccharides; or    -   (e) any combination of (a) through (d).

Examples of said carboxylic acid ester substrate may include monoacetin;triacetin; monopropionin; dipropionin; tripropionin; monobutyrin;dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate;acetylated xylan; acetylated xylan fragments;β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal;tri-O-acetyl-glucal; propylene glycol diacetate; ethylene glycoldiacetate; monoesters or diesters of 1,2-ethanediol; 1,2-propanediol;1,3-propanediol; 1,2-butanediol; 1,3-butanediol; 2,3-butanediol;1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol; 1,6-pentanediol,1,2-hexanediol; 2,5-hexanediol; 1,6-hexanediol; or any combinationthereof.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane, 1,2,3-propanetriol triacetate and all othersynonyms of GAS Registry Number 102-76-1.

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of GASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 13945-7.

As used herein, the term “ethyl acetate” is synonymous with aceticether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoicacid ethyl ester, ethyl acetic ester and all other synonyms of CASRegistry Number 141-78-6.

As used herein, the term “ethyl lactate” is synonymous with lactic acidethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms “acetylated sugar” and “acetylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acetylgroup. Examples include, but are not limited to, glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” is meant a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyctopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety isa straight chain, branched or cyclic arrangement of carbon atomsconnected by single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol,1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,2-pentanedial, 2,5-pentanediol,1,6-pentanedial, 1,2-hexanediol, 2,5-hexanediol, 1,6-hexanediol, andmixtures thereof refer to said compounds comprising at least one estergroup of the formula RC(O)O, wherein R is a C1 to C7 linear hydrocarbylmoiety. In one embodiment, the carboxylic acid ester substrate comprisespropylene glycol diacetate (PGDA), ethylene glycol diacetate (EDGA) ormixtures thereof.

As used herein, the term “propylene glycol diacetate” is synonymous with1,2-diacetoxypropane, propylene diacetate, 1,2-propanediol diacetate,and all other synonyms of CAS Registry Number 623-84-7.

As used herein, the term “ethylene glycol diacetate” is synonymous with1,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all othersynonyms of CAS Registry Number 111-55-7.

As used herein, the terms “suitable enzymatic reaction mixture”,“components suitable for in situ generation of a peracid”, “suitablereaction components”, “suitable aqueous reaction mixture”, and “reactionmixture” refer to the materials and water in which the reactants andenzyme catalyst come into contact. The components of the suitableaqueous reaction mixture are provided herein and those skilled in theart appreciate the range of component variations suitable for thisprocess. In one embodiment, the suitable enzymatic reaction mixtureproduces peracid in situ upon combining the reaction components. Assuch, the reaction components may be provided as a multi-componentsystem wherein the reaction components remains separated until use. Thedesign of systems and means for separating and combining multiple activecomponents are known in the art and generally will depend upon thephysical form of the individual reaction components. For example,multiple active fluids (liquid-liquid) systems typically usemultichamber dispenser bottles or two-phase systems (e.g., U.S. PatentApplication Publication No. 2005/0139608; U.S. Pat. No. 5,398,846; U.S.Pat. No. 5,624,634; U.S. Pat. No. 6,391,840; E.P. Patent 0807156B1; U.S.Patent Application Publication No. 2005/0008526; and PCT Publication No.WO 00/61713) such as found in some bleaching applications wherein thedesired bleaching agent is produced upon mixing the reactive fluids.Other forms of multi-component systems used to generate peracid mayinclude, but are not limited to, those designed for one or more solidcomponents or combinations of solid-liquid components, such as powders(e.g., U.S. Pat. No. 5,116,575), multi-layered tablets (e.g., U.S. Pat.No. 6,210,639), water dissolvable packets having multiple compartments(e.g., U.S. Pat. No. 6,995,125) and solid agglomerates that react uponthe addition of water (e.g., U.S. Pat. No. 6,319,888).

In multi-component systems, active constituents are initially separatedfrom each other in one or more respective components and then combinedto form a reaction formulation. A multi-component system can face suchproblems as the failure of the active components to satisfactorilycombine, the neutralization or reduction of the activity of one or morecomponents, and/or the formation of a reaction formulation that is notcompatible with delivery requirements. For example, in a multi-componentsystem including at least one component comprising an enzyme and asubstrate for such enzyme that is insoluble or partially insoluble in asecond component comprising water, at least three conditions may arisethat interfere with the ability to efficaciously produce and deliver aperoxycarboxylic acid product: first, the viscosity of theenzyme/substrate constituent can be too high to permit efficient mixingwith a second constituent comprising a source of peroxygen, whichdecreases the rate of production of peroxycarboxylic acid; second, theviscosity of the enzyme/substrate constituent can be too high to permitcertain modes of delivery of a product comprising a mixture of theenzyme/substrate constituent and the source of peroxygen, such asspraying; third, the dissolution rate of the substrate in theenzyme/substrate component after mixing with a second componentcomprising a source of peroxygen in aqueous solution is too low topermit a satisfactory rate of production of peroxycarboxylic acid. Theseproblems also become evident in situations where use of a particularratio of a component comprising an aqueous source of peroxygen to acomponent comprising an enzyme/substrate constituent is desired.

It is well known to those skilled in the art that organic solvents canbe deleterious to the activity of enzymes, either when enzymes aresuspended directly in organic solvents, or when miscible organic/aqueoussingle phase solvents are employed. Two literature publications thatreview the effects of organic solvents on enzyme activity and structureare: (a) C. Laane et al., supra and (b) D. A. Cowan and A. R. Plant,supra. Cowan and Plant note (on page 87) that the art generallyrecognizes that there is little or no value in using organic solventshaving a log P≦2 to stabilize intracellular enzymes in an organic phasesystem, where log P is defined as the logarithm of the partitioncoefficient of a substance between octanol and water, expressed asP=[solute]_(octanol)/[solute]_(water). Organic solvents having a log Pbetween 2 and 4 can be used on a case-by-case basis dependent on enzymestability, and those having a log P>4 are generally useful in organicphase systems.

Cowan and Plant further note (on page 91) that the effect of directexposure of an enzyme dissolved in a single-phase organic-aqueoussolvent depends on solvent concentration, solvent/enzyme surface groupinteractions, and solvent/enzyme hydration shell interactions. Because asolvent's log P must be sufficiently low so that the solvent is fullymiscible with the aqueous phase to produce a single-phase, asingle-phase organic-aqueous solvent containing a low log P organicsolvent usually has a negative effect on enzyme stability except in loworganic solvent concentration applications. Thus, organic solventshaving a low log P are traditionally thought to be detrimental to enzymestability at anything but unworkably low concentrations.

Triacetin is reported to have a log P of 0.25 (Y. M. Gunning, et al., J.Agric. Food Chem. 48:395-399 (2000)), similar to that of ethanol (log P−0.26) and isopropanol (log P 0.15) (Cowan and Plant); therefore thestorage of enzyme powder in triacetin would be expected to result inunacceptable loss of enzyme activity, as would the use of additionalcosolvents with log P<2 (e.g., cyclohexanone, log P=0.94) (Cowan andPlant); 1,2-propanediol, log P=−1.41 (Gunning, et al.); 1,3-propanediol,log P=−1.3 (S-J, Kuo, et al., J. Am. Oil Chem. Soc. 73:1427-1433 (1996);diethylene glycol butyl ether, log P=0.56 (N. Funasaki, et al., J. Phys.Chem. 68:5786-5790 (1984); triethyleneglycol, log P=−1.75 (L. Braeken,et al., ChemPhysChem 6:1606-1612 (2005)). Applying the above-referencedteachings of Cowan and Plant, it would be expected that the solventslisted above, having low log P values, could not suitably be included inan enzyme-containing first component of a multicomponent system withoutinactivation of the enzyme (either prior to or after mixing of the firstcomponent with a second component comprising a source of peroxygen inwater).

However, it has surprisingly been discovered that the inclusion of acosolvent comprising an organic solvent having a log P of less thanabout 2 may function to aid in the dissolution of enzyme substrate withpoor solubility in water and/or may function as a diluent of Component Ato enable mixing of a desired ratio of Components A and B. In otherwords, the cosolvent may resolve conditions (e.g., unacceptably highviscosity of an enzyme/substrate component, poor mixing of thiscomponent with a source of peroxygen in water) that otherwise interferewith the ability to efficaciously produce and deliver a peroxycarboxylicacid product in a form that can be delivered by means that areconventional to (or otherwise suitable in) consumer, industrial, andmedical contexts.

In the methods and systems described herein, the cosolvent comprising anorganic solvent having a log P of less than about 2, wherein log P isdefined as the logarithm of the partition coefficient of a substancebetween octanol and water, expressed asP=[solute]_(octanol)/[solute]_(water), solubilizes the substrate in theaqueous reaction formulation without substantial loss of perhydrolyticactivity of the enzyme catalyst; wherein the cosolvent is not asubstrate for said enzyme catalyst.

In some embodiments, the first component comprising the formulation ofthe enzyme catalyst and the carboxylic acid ester substrate optionallycomprises an inorganic or organic buffer, a stabilizer, a corrosioninhibitor, a wetting agent, or combinations thereof. In someembodiments, the source of peroxygen comprises a hydrogen peroxidestabilizer.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with peroxide to form a peracid. Typically, inorganicperoxide is reacted with the selected substrate in the presence of acatalyst to produce the peracid. As used herein, the term “chemicalperhydrolysis” includes perhydrolysis reactions in which a substrate(i.e., a peracid precursor) is combined with a source of hydrogenperoxide wherein peracid is formed in the absence of an enzyme catalyst.

As used herein, the term “perhydrolase activity” refers to the catalystactivity per unit mass (for example, milligram) of protein, dry cellweight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmol of peracid product per minute at a specifiedtemperature.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme having perhydrolysis activityand may be in the form of a whole microbial cell, permeabilizedmicrobial cell(s), one or more cell components of a microbial cellextract, partially purified enzyme, or purified enzyme. The enzymecatalyst may also be chemically modified (e.g., by pegyfation or byreaction with cross-linking reagents). The perhydrolase catalyst mayalso be immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells; Gordon F, Bickerstaff, Editor, Humana Press,Totowa, N.J., USA; 1997. As described herein, all of the present enzymeshaving perhydrolysis activity are structurally members of thecarbohydrate esterase family 7 (“CE-7” family) of enzymes (see Coutinho,P. M., Henrissat, B. “Carbohydrate-active enzymes: an integrateddatabase approach” in Recent Advances in Carbohydrate Bioengineering, H.J. Gilbert, G. Davies, B, Henrissat and B. Svensson eds., (1999) TheRoyal Society of Chemistry, Cambridge, pp. 3-12). The CE-7 family ofenzymes has been demonstrated to be particularly effective for producingperacids from a variety of carboxylic acid ester substrates whencombined with a source of peroxygen (See PCT publication No.WO2007/070609 and U.S. Patent Application Publication Nos. 2008/0176299,2008/176783, and 2009/0005590 to DiCosimo at al.; each hereinincorporated by reference in their entireties).

Members of the CE-7 family include cephalosporin C deacetylases (CAHs;E.C. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72), Membersof the CE-7 esterase family share a conserved signature motif (Vincentet al., supra). Perhydrolases comprising the CE-7 signature motif and/ora substantially similar structure are suitable for use in the presentinvention. Means to identify substantially similar biological moleculesare well known in the art (e.g., sequence alignment protocols, nucleicacid hybridizations, and/or presence of a conserved signature motif). Inone aspect, the enzyme catalyst in the present processes and systemscomprises a substantially similar enzyme having at feast 30%, preferablyat least 33%, more preferably at least 40%, more preferably at least50%, even more preferably at least 60%, yet even more preferable atleast 70%, yet even more preferably at least 80%, yet even morepreferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%amino acid identity to the sequences provided herein. The nucleic acidmolecules encoding the present CE-7 carbohydrate esterases are alsoprovided herein. In further embodiments, the perhydrolase catalystuseful in the present processes and systems is encoded by a nucleic acidmolecule that hybridizes under highly stringent conditions to one of thepresent nucleic acid molecules.

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., (1995) Appl. Env.Microbiol. 61(6):2224-2229). As described herein, several cephalosporinC deacetylases are provided having significant perhydrolysis activity.

As used herein, “acetyl xylan esterases” refers to an enzyme (E.C.3.1.172; AXEs) that catalyzes the deacetylation of acetylated xylans andother acetylated saccharides. As illustrated herein, several enzymesclassified as acetyl xylan esterases are provided having significantperhydrolase activity.

As used herein, the term “Bacillus subtilis ATCC® 31954™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC®)having international depository accession number ATCC® 31954™. Bacillussubtilis ATCC® 31954™ has been reported to have an ester hydrolase(“diacetinase”) activity capable of hydrolyzing glycerol esters having2-carbon to 8-carbon acyl groups, especially diacetin (U.S. Pat. No.4,444,886; herein incorporated by reference in its entirety). Asdescribed herein, an enzyme having significant perhydrolase activity hasbeen isolated from B. subtilis ATCC® 31954™ and is provided as SEQ. IDNO: 2. The amino acid sequence of the isolated enzyme has 100% aminoacid identity to the cephalosporin C deacetylase provided by GENBANK®Accession No, BAA01729.1 (Mitsushima et al., supra).

As used herein, the term “Bacillus subtilis BE1010” refers to the strainof Bacillus subtilis as reported by Payne and Jackson (J. Bacteriol.173:2278-2282 (1991)). Bacillus subtilis BE101D is a derivative ofBacillus subtilis subsp. subtilis strain BR151 (ATCC® 33677™) having achromosomal deletion in the genes encoding subtilisin and neutralprotease. As described herein, an enzyme having significant perhydrolaseactivity has been isolated from B. subtilis BE1010 and is provided asSEQ ID NO: 4. The amino acid sequence of the isolated enzyme has 100%amino acid identity to the cephalosporin deacetylase reported inBacillus subtilis subsp. subtilis strain 168 (Kunst et al., Nature390:249-256 (1997)).

As used herein, the term “Bacillus subtilis ATCC® 29233™” refers to astrain of Bacillus subtilis deposited to the American Type CultureCollection (ATCC®) having international depository accession numberATCC® 29233™. As described herein, an enzyme having significantperhydrolase activity has been isolated and sequenced from B. subtilisATCC® 29233™ and is provided as SEC) ID NO: 26.

As used herein, the term “Clostridium thermocellum ATCC® 27405™” refersto a strain of Clostridium thermocellum deposited to the American TypeCulture Collection (ATCC®) having international depository accessionnumber ATCC® 27405™. The amino acid sequence of the enzyme havingperhydrolase activity from C. thermocellum ATCC® 27405™ is provided asSEQ ID NO: 12.

As used herein, the term “Bacillus subtilis ATCC® 6633™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC®)having international depository accession number ATCC® 6633™. Bacillussubtilis ATCC® 6633™ has been reported to have cephalosporinacetylhydrolase activity (U.S. Pat. No. 6,465,233). The amino acidsequence of the enzyme having perhydrolase activity from B. subtilisATCC® 6633™ is provided as SEQ ID NO: 5.

As used herein, the term “Bacillus licheniformis ATCC®14580™” refers toa bacterial cell deposited to the American Type Culture Collection(ATCC®) having international depository accession number ATCC® 14580™.Bacillus licheniformis ATCC® 14580™ has been reported to havecephalosporin acetylhydrolase activity (GENBANK® YP_(—)077621). Theamino acid sequence of the enzyme having perhydrolase activity from B.licheniformis ATCC® 14580™ is provided as SEQ ID NO: 8.

As used herein, the term “Bacillus pumilus PS213” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®AJ249957). The amino acid sequence of the enzyme having perhydrolaseactivity from Bacillus pumilus PS213 is provided as SEQ ID NO: 10.

As used herein, the term “Thermotoga neapolitana” refers to a strain ofThermotoga neapolitana reported to have acetyl xylan esterase activity(GENBANK® AAB70869). The amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga neapolitana is provided as SEQ IDNO: 14. A variant enzyme derived from SEQ ID NO: 14 has recently beenreported having improved perhydrolysis activity and is provided as SEQID NO: 73 (See co-owned, co-filed, and copending U.S. patent applicationAttorney Docket No. CL4392 US NA entitled “IMPROVED PERHYDROLASES FORENZYMATIC PERACID GENERATION”; incorporated herein by reference in itsentirety), where the Xaa residue at position 277 is Ala, Val, Ser, orThr.

As used herein, the term “Thermotoga maritima MSB8” refers to a strainof Thermotoga maritima reported to have acetyl xylan esterase activity(GENBANK® NP_(—)227893.1). The amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga maritima is provided as SEQ ID NO;16. A variant enzyme derived from SEQ ID NO: 16 has recently beenreported having improved perhydrolysis activity and is provided as SEQID NO: 74 (See co-owned, co-filed, and copending U.S. patent applicationAttorney Docket No. CL4392 US NA), where the Xaa residue at position 277is Ala, Val, Ser, or Thr.

As used herein, the term “Bacillus clausii KSM-K16” refers to abacterial cell reported to have cephalosporin-C deacetylase activity(GENBANK® YP_(—)175265). The amino acid sequence of the enzyme havingperhydrolase activity from Bacillus clausii KSM-K16 is provided as SEQID NO: 24.

As used herein, the term “Thermoanearobacterium saccharolyticum” refersto a bacterial strain reported to have acetyl xylan esterase activity(GENBANK® S41858). The amino acid sequence of the enzyme havingperhydrolase activity from Thermoanearobacterium saccharolyticum isprovided as SEQ ID NO: 54.

As used herein, the term “Thermotoga lettingae” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®CP000812). The deduced amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga lettingae is provided as SEQ IDNO: 60.

As used herein, the term “Thermotoga petrophila” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®CP000702). The deduced amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga lettingae is provided as SEQ IDNO: 64.

As used herein, the term “Thermotoga sp. RQ2” refers to a bacterial cellreported to have acetyl xylan esterase activity (GENBANK® CP000969). Twodifferent acetyl xylan esterases have been identified from Thermotoga spRQ2 and are referred to herein as “RQ2(a)” (the deduced amino acidsequence provided as SEQ ID NO: 68) and “RQ2(b)” (the deduced amino acidsequence provided as SEQ ID NO: 72).

As used herein, an “isolated nucleic acid molecule” and “isolatednucleic acid fragment” will be used interchangeably and refer to apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid molecule in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid Xaa X (or asdefined herein)

As used herein, “substantially similar” refers to nucleic acid moleculeswherein changes in one or more nucleotide bases results in the addition,substitution, or deletion of one or more amino acids, but does notaffect the functional properties (i.e., perhydrolytic activity) of theprotein encoded by the DNA sequence. As used herein, “substantiallysimilar” also refers to an enzyme having an amino acid sequence that isat least 30%, preferably at least 33%, more preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, evenmore preferably at least 70%, even more preferably at least 80%, yeteven more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the sequences reported herein wherein theresulting enzyme retains the present functional properties (i.e.,perhydrolytic activity). “Substantially similar” may also refer to anenzyme having perhydrolytic activity encoded by nucleic acid moleculesthat hybridize under highly stringent conditions to the nucleic acidmolecules reported herein. It is therefore understood that the inventionencompasses more than the specific exemplary sequences.

For example, it is well known in the art that alterations in a genewhich result in the production of a chemically equivalent amino acid ata given site, but do not affect the functional properties of the encodedprotein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly);    -   2, Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   3. Polar, positively charged residues: His, Arg, Lys;    -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);        and    -   5. Large aromatic residues: Phe, Tyr, and Trp.        Thus, a codon for the amino acid alanine, a hydrophobic amino        acid, may be substituted by a codon encoding another less        hydrophobic residue (such as glycine) or a more hydrophobic        residue (such as valine, leucine, or isoleucine). Similarly,        changes which result in substitution of one negatively charged        residue for another (such as aspartic acid for glutamic acid) or        one positively charged residue for another (such as lysine for        arginine) can also be expected to produce a functionally        equivalent product. In many cases, nucleotide changes which        result in alteration of the N-terminal and C-terminal portions        of the protein molecule would also not be expected to alter the        activity of the protein.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of biological activity of theencoded products. Moreover, the skilled artisan recognizes thatsubstantially similar sequences are encompassed by the presentinvention. In one embodiment, substantially similar sequences aredefined by their ability to hybridize, under highly stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS, 65° C.) with the sequences exemplified herein. Insome embodiments, the present methods and systems may include enzymeshaving perhydrolase activity encoded by isolated nucleic acid moleculesthat hybridize under stringent conditions to the nucleic acid moleculesreported herein. In preferred embodiments, the present methods andsystems employ an enzyme having perhydrolase activity encoded byisolated nucleic acid molecule that hybridize under stringent conditionsto a nucleic acid molecule having a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5; SEQ IDNO: 7; SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO: 25; SEQ IDNO:29; SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69,SEQ ID NO: 70, and SEQ ID NO: 71.

As used herein, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle strand of the first molecule can anneal to the other moleculeunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook, J, and Russell, D., T. Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(2001). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjustedto screen for moderately similar molecules, such as homologous sequencesfrom distantly related organisms, to highly similar molecules, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes typically determine stringency conditions. Oneset of preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of stringent hybridizationconditions is 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDSfollowed by a final wash of 0.1×SSC, 0.1% SDS, 65° C. with the sequencesexemplified herein.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrook andRussell, supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrookand Russell, supra). In one aspect, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Preferably, a minimumlength for a hybridizable nucleic acid is at least about 15 nucleotidesin length, more preferably at least about 20 nucleotides in length, evenmore preferably at least 30 nucleotides in length, even more preferablyat least 300 nucleotides in length, and most preferably at least 800nucleotides in length. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

As used herein, the term “percent identity” is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.), the AlignX program of Vector NTI v.7.0 (Informax, Inc.,Bethesda, Md.), or the EMBOSS Open Software Suite (EMBL-EBI; Rice etal., Trends in Genetics 16, (6) pp 276-277 (2000)). Multiple alignmentof the sequences can be performed using the CLUSTAL method (such asCLUSTALW; for example version 1.83) of alignment (Higgins and Sharp,CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res.22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res 31(13):3497-500 (2003)), available from the European Molecular BiologyLaboratory via the European Bioinformatics Institute) with the defaultparameters. Suitable parameters for CLUSTALW protein alignments includeGAP Existence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g.,Gonnet250), protein ENDGAP=−1, Protein GAPDIST=4, and KTUPLE=1. In oneembodiment, a fast or slow alignment is used with the default settingswherein a slow alignment is preferred. Alternatively, the parametersusing the CLUSTALW method (version 1.83) may be modified to also useKTUPLE=1, GAP PENALTY=10, GAP extension=1, matrix=BLOSUM (e.g.BLOSUM64), WINDOW=5, and TOP DIAGONALS SAVED=5.

In one aspect of the present methods and systems, suitable isolatednucleic acid molecules (isolated polynucleotides of the presentinvention) encode a polypeptide having an amino acid sequence that is atleast about 30%, preferably at least 33%, preferably at least 40%,preferably at least 50%, preferably at least 60%, more preferably atleast 70%, more preferably at feast 80%, even more preferably at least85%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequences reported herein.Suitable nucleic acid molecules of the present invention not only havethe above homologies, but also typically encode a polypeptide havingabout 300 to about 340 amino adds, more preferably about 310 to about330 amino acids, and most preferably about 318 to about 325 amino acidsin length.

As used herein, the terms “signature motif” and “diagnostic motif” referto conserved structures shared among a family of enzymes having adefined activity. The signature motif can be used to define and/oridentify the family of structurally related enzymes having similarenzymatic activity for a defined family of substrates. The signaturemotif can be a single contiguous amino acid sequence or a collection ofdiscontiguous, conserved motifs that together form the signature motif.Typically, the conserved motif(s) is represented by an amino acidsequence. As described herein, the present perhydrolases belong to thefamily of CE-7 carbohydrate esterases. This family of enzymes can bedefined by the presence of a CE-7 “signature motif” (Vincent et al.,supra).

As used herein, “codon degeneracy” refers to the nature of the geneticcode permitting variation of the nucleotide sequence without affectingthe amino acid sequence of an encoded polypeptide. Accordingly, thepresent invention relates to any nucleic acid molecule that encodes allor a substantial portion of the amino acid sequences encoding thepresent microbial polypeptide. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

As used herein, the term “codon optimized”, as it refers to genes orcoding regions of nucleic acid molecules for transformation of varioushosts, refers to the alteration of codons in the gene or coding regionsof the nucleic acid molecules to reflect the typical codon usage of thehost organism without altering the polypeptide for which the DNA codes.

As used herein, “synthetic genes” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form gene segments that are then enzymatically assembled toconstruct the entire gene. “Chemically synthesized”, as pertaining to aDNA sequence, means that the component nucleotides were assembled invitro. Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the genes can be tailored for optimal gene expression basedon optimization of nucleotide sequences to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

As used herein, “gene” refers to a nucleic acid molecule that expressesa specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different from that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Suitable regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence, Regulatorysequences may include promoters, translation leader sequences, RNAprocessing site, effector binding site and stem-loop structure.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene at different stages of development, or in responseto different environmental or physiological conditions. Promoters thatcause a gene to be expressed at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of different lengths may haveidentical promoter activity.

As used herein, the “3′ non-coding sequences” refer to DNA sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences (normally limited to eukaryotes) and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts(normally limited to eukaryotes) to the 3′ end of the mRNA precursor.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence, i.e., that the coding sequenceis under the transcriptional control of the promoter. Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, the term “expression” refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid molecule described herein. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein, “transformation” refers to the transfer of a nucleicacid molecule into the genome of a host organism, resulting ingenetically stable inheritance. In the present invention, the hostcell's genome includes chromosomal and extrachromosomal plasmid) genes.Host organisms containing the transformed nucleic acid molecules arereferred to as “transgenic” or “recombinant” or “transformed” organisms.

As used herein, the terms “plasmid”, “vector” and “cassette” refer to anextrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein, the term “sequence analysis software” refers to anycomputer algorithm or software program that is useful for the analysisof nucleotide or amino acid sequences. “Sequence analysis software” maybe commercially available or independently developed. Typical sequenceanalysis software will include, but is not limited to, the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), CLUSTALW (for example, version 1.83; Thompson et al.,Nucleic Acids Research, 22(22):4673-4680 (1994), and the FASTA programincorporating the Smith-Waterman algorithm (W. R. Pearson, Cornput.Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,111-20. Editor(s): Suhai, Sander. Publisher: Plenum, New York, N.Y.),Vector NTI (Informax, Bethesda, Md.) and Sequencher v. 4.05. Within thecontext of this application it will be understood that where sequenceanalysis software is used for analysis, that the results of the analysiswill be based on the “default values” of the program referenced, unlessotherwise specified. As used herein “default values” will mean any setof values or parameters set by the software manufacturer that originallyload with the software when first initialized.

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to, microorganisms, spores, viruses, prions, and mixturesthereof. The process produces an efficacious concentration of at leastone percarboxylic acid useful to reduce and/or eliminate the presence ofthe viable biological contaminants. In a preferred embodiment, thebiological contaminant is a viable pathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. As used herein, the term “disinfection” refersto the act or process of disinfecting. Typically, disinfectants are usedto treat inanimate objects or surfaces. As used herein, the term“antiseptic” refers to a chemical agent that inhibits the growth ofdisease-carrying biological contaminants (such as microorganisms). Inone aspect of the present methods and systems, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “sanitary” means of or relating to therestoration or preservation of health, typically by removing, preventingor controlling an agent that may be injurious to health. As used herein,the term “sanitize” means to make sanitary. As used herein, the term“sanitizes” refers to a sanitizing agent. As used herein the term“sanitization” refers to act or process of sanitizing.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peracids can have virucidal activity.Typical alternative virucides known in the art which may be suitable foruse with the present invention include, for example, alcohols, ethers,chloroform, formaldehyde, phenols, beta propiolactone, iodine, chlorine,mercury salts, hydroxylamine, ethylene oxide, ethylene glycol,quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peracids can have biocidalactivity. Typical alternative biocides known in the art, which may besuitable for use in the present invention include, for example,chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone,acrolein, amines, chlorinated phenolics, copper salts, organo-sulphurcompounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3-log reduction, morepreferably at least a 4-log reduction, and most preferably at least a5-log reduction. In another aspect, the minimum biocidal concentrationis at least a 6-log reduction in viable microbial cells.

As used herein, the term “benefit agent” refers to something thatpromotes or enhances a useful advantage or favorable effect. In oneembodiment, methods and systems are provided whereby a benefit agent,such as a composition comprising a peroxycarboxylic acid, is applied toan article of clothing or textile to achieve a desired benefit, such asdisinfecting, sanitizing, bleaching, stain removal, deodorizing/odorreduction, and any combination thereof.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates. As described herein, the concentration ofthe hydrogen peroxide provided by the peroxygen compound in the aqueousreaction formulation is initially at least 1 mM or more upon combiningthe reaction components. In one embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is at least 10 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction formulation is at least 100 mM. In another embodiment, thehydrogen peroxide concentration in the aqueous reaction formulation isat least 200 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is 500 mM or more. Inyet another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is 1000 mM or more. The molar ratio of thehydrogen peroxide to enzyme substrate, e.g. triglyceride,(H₂O₂:substrate) in the aqueous reaction formulation may be from about0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5to 5.

In some embodiments of the presently disclosed methods and systems, theenzyme catalyst comprises a perhydrolase having a structure belonging tothe CE-7 carbohydrate esterase family. In other embodiments, theperhydrolase catalyst is structurally classified as a cephalosporin Cdeacetylase. In other embodiments, the perhydrolase catalyst isstructurally classified as an acetyl xylan esterase. The terms “enzymecatalyst”, “enzyme catalyst having perhydrolysis activity”, and“perhydrolase catalyst” are used herein interchangeably.

In some embodiments of the present methods and systems, the perhydrolasecatalyst comprises an enzyme having a CE-7 signature motif that alignswith a reference sequence SEQ ID NO: 2 using CLUSTALW, said CE-7signature motif comprising:

i) an RGQ motif at amino acid positions 118-120 of SEQ ID NO:2;

ii) a GXSQG motif at amino acid positions 179-183 of SEQ ID NO:2; and

iii) an HE motif at amino acid positions 298-299 of SEQ ID NO:2;

wherein said enzyme also comprises at least 30% amino acid identity toSEQ ID NO:2.

In other embodiments of the present methods and systems, the signaturemotif additional comprises a fourth conserved motif defined as an LXDmotif at amino acid residues 267-269 when aligned to reference sequenceSEQ ID NO: 2 using CLUSTALW.

In additional embodiments of the present methods and systems, theperhydrolase catalyst may comprise an enzyme having perhydrolaseactivity selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 64, SEQID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74 or asubstantially similar enzyme having perhydrolase activity derived bysubstituting, deleting or adding one or more amino acids to said aminoacid sequence.

In other embodiments of the present methods and systems, substantiallysimilar enzyme having perhydrolase activity is at least 30%, preferablyat least 33%, more preferably at least 40%, more preferably at least50%, even more preferably at least 60%, yet even more preferable atleast 70%, yet even more preferably at least 80%, yet even morepreferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to one or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 54, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQID NO: 73, and SEQ ID NO: 74.

In other embodiments of the present methods and systems, theperhydrolase catalyst comprises an enzyme having an amino acid sequenceencoded by a nucleic acid molecule that hybridizes to a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO: 25; SEQ ID NO:29; SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71 under stringenthybridization conditions.

In other embodiments of the present methods and systems, theperhydrolase catalyst comprises an enzyme having an amino acid sequenceselected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 73(i.e., the wild type Thermotoga neapolitana and Thermotoga neapolitanavariants having an amino acid substitution at amino acid residue 277).

In other embodiments of the present methods and systems, theperhydrolase catalyst comprises an enzyme having an amino acid sequenceselected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 74(i.e., the wild type Thermotoga maritima and Thermotoga maritimavariants having an amino acid substitution at amino acid residue 277).

In other embodiments of the present methods and systems, theperhydrolase catalyst comprises an enzyme having at least 30%,preferably at least 36%, amino acid identity to a contiguous signaturemotif defined as SEQ ID NO: 49 wherein the conserved motifs describedabove (i.e., RGQ, GXSQG, and HE, and optionally, LXD) are conserved.

With respect to the presently disclosed methods and systems, suitablecarboxylic acid ester substrates include esters provided by thefollowing formula:[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O

R₆=C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionallysubstituted with hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆optionally comprises one or more ether linkages for R₆=C2 to C7;

R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionallysubstituted with hydroxyl groups: wherein each carbon atom in R₅individually comprises no more than one hydroxyl group or no more thanone ester group; wherein R₅ optionally comprises one or more etherlinkages;

m=1 to the number of carbon atoms in R₅ ⁻, and

wherein said esters have solubility in water of at least 5 ppm at 25° C.

In other embodiments of the present methods and systems, suitablesubstrates also include esters of the formula:

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with a hydroxyl or a C1 to C4 alkoxy group and R₂=C1 to C10straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or(CH₂CH(CH₃)—O)_(n)H and n=1 to 10.

In other embodiments of the present methods and systems, suitablecarboxylic acid ester substrates include glycerides of the formula:

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with a hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄ areindividually H or R₁C(O).

In other embodiments of the present methods and systems, R₆ is C1 to C7linear hydrocarbyl moiety, optionally substituted with hydroxyl groupsor C1 to C4 alkoxy groups, optionally comprising one or more etherlinkages. In further preferred embodiments, R₆ is C2 to C7 linearhydrocarbyl moiety, optionally substituted with hydroxyl groups, and/oroptionally comprising one or more ether linkages.

In other embodiments of the disclosed methods and systems, suitablecarboxylic acid ester substrates also include acetylated saccharidesselected from the group consisting of acetylated mono-, di-, andpolysaccharides. In preferred embodiments, the acetylated saccharidesinclude acetylated mono-, di-, and polysaccharides. In otherembodiments, the acetylated saccharides are selected from the groupconsisting of acetylated xylan, fragments of acetylated xylan,acetylated xylose (such as xylose tetraacetate), acetylated glucose(such as glucose pentaacetate), β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetylatedcellulose. In preferred embodiments, the acetylated saccharide isselected from the group consisting ofβ-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-D-glucal, and acetylated cellulose. As such, acetylatedcarbohydrates may be suitable substrates for generating percarboxylicacids using the present methods and systems (i.e., in the presence of aperoxygen source).

In additional embodiments of the present methods and systems, thecarboxylic acid ester substrate may be monoacetin; triacetin;monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin;tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan;acetylated xylan fragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-glucal; propylene glycoldiacetate; ethylene glycol diacetate; monoesters or diesters of1,2-ethanediol; 1,2-propanediol; 1,3-propanediol; 1,2-butanediol;1,3-butanediol; 2,3-butanediol; 1,4-butanediol; 1,2-pentanediol;2,5-pentanediol; 1,6-pentanediol; 1,2-hexanediol; 2,5-hexanediol;1,6-hexanediol; and mixtures thereof. In preferred embodiments of thepresent methods and systems, the substrate comprises triacetin.

Preferably, the substrate used in the present methods and systems hassolubility in water of less than about 100 mg/mL. In some embodiments ofthe present methods and systems, the percent by weight of the substratein the aqueous reaction mixture exceeds the solubility limit of thesubstrate in water.

In other embodiments of the presently disclosed methods and systems, thesubstrate may be ethyl acetate; methyl lactate; ethyl lactate; methylglycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate;methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; triethyl 2-acetylcitrate; glucose pentaacetate; gluconolactone; glycerides (mono-, di-,and triglycerides) such as monoacetin, diacetin, triacetin,monopropionin, dipropionin (glyceryl dipropionate), tripropionin(1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryldibutyrate), tributyrin (1,2,3-tributyrylglycerol); acetylatedsaccharides; and mixtures thereof.

In a further preferred aspect of the present methods and systems, thecarboxylic acid ester substrates are selected from the group consistingof monoacetin, diacetin, triacetin, monopropionin, dipropionin,tripropionin, monobutyrin, dibutyrin, tributyrin, ethyl acetate, andethyl lactate. In yet another aspect, the carboxylic acid estersubstrates are selected from the group consisting of diacetin,triacetin, ethyl acetate, and ethyl lactate. In preferred aspects, thecarboxylic acid ester is a glyceride selected from the group consistingof monoacetin, diacetin, triacetin, and mixtures thereof.

Use of Cosolvents in a Multi-Component Generation System.

The cosolvent for use in the presently disclosed methods and systemscomprises an organic solvent having a log P of less than about 2. Insome embodiments, the cosolvent is preferably an alcohol. The cosolventpreferably may comprise tripropylene glycol methyl ether, dipropyleneglycol methyl ether, propylene glycol methyl ether, diethylene glycolbutyl ether, dipropylene triethyleneglycol, 1,2-propanediol,N-ethyl-2-pyrroldinone, isopropanol, ethanol, ethyl lactate,1,3-propanediol, or any combination thereof. In some embodiments of thepresent methods and systems, the cosolvent comprises tripropylene glycolmethyl ether.

In the present methods for producing peroxycarboxylic acid in amulticomponent system, the multicomponent system may comprise a firstcomponent comprising the catalyst composition comprising a cosolvent,and a second component comprising the source of peroxygen and water. Thesecond component may comprise an optional stabilizer to extend theshelf-life of the hydrogen peroxide. The catalyst, substrate, and thecosolvent may be combined to form the first component prior tocombination of the first component with the second component. Likewise,with respect to the disclosed methods for disinfecting a surface, thestep of combining the catalyst composition and the source of peroxygenmay comprise combining a first component comprising the catalystcomposition and the cosolvent with a second component comprising thesource of peroxygen and water.

In embodiments of the present methods and systems wherein themulticomponent system comprises a first component comprising thecatalyst composition comprising the cosolvent, and a second componentcomprising the source of peroxygen and water, the first component may becombined with the second component in a ratio of about 1:1 to about 1:10by weight. In one embodiment, the first component is combined with thesecond component in a ratio of about 1:9 by weight.

Within the range of a 1:1 to a 1:10 mixture of the first and secondcomponents as described above, a concentration of enzyme substrate(“activator”) in the first component and a ratio of the second componentto the first component are also chosen such that the substrate issoluble in the final mixture of the first component and the secondcomponent. For example, the solubility of triacetin in water is reportedas 71.7 g/L (Seelig, Chemische Berichte; 24: 3466 (1891)), thesolubility of 1,2-propanediol diacetate is reported to be 1 part in 10parts water (Wurtz, Annales de Chimie; 55:443 (1859); Justus LiebigsAnnalen der Chemie; 105:206 (1858)), and the solubility of tributyrin inwater is reported to be 0.015% (volume/volume) (Loskit, Zeitschrift fuerPhysikalische Chemie, Stoechiometrie and Verwandtschaftslehre; 134:137(1928)). Given the wide range of solubilities for the enzyme substratesof the present invention, where the enzyme substrates are describedbelow, the cosolvent added to the first component of the presentinvention may function to aid in the dissolution of an enzyme substratewith poor solubility in water, in addition to its function as a diluentin the first component to enable the mixing of a desired ratio of thefirst component and the second component. In one embodiment of theinvention, the substrate is soluble in the resulting formulation of thefirst component and the second component at a concentration of at least25% (weight/weight). In a second embodiment of the present invention,the substrate is soluble in the resulting formulation of the firstcomponent and the second component at a concentration of at least 10%(weight/weight). In a third embodiment of the present invention, thesubstrate is soluble in the resulting formulation of the first componentand the second component at a concentration of at least 5%(weight/weight). In a fourth embodiment of the present invention, thesubstrate is soluble in the resulting formulation of the first componentand the second component at a concentration of at least 2%(weight/weight).

In one embodiment, the cosolvent may be present in the first componentin an amount of about 20% to about 80% by weight. The substrate may bepresent in the first component in an amount of about 10% to about 60% byweight. In some embodiments, the first component may comprise about 55%by weight substrate, about 40% by weight cosolvent, about 0.3% by weightenzyme catalyst, and about 2.5% by weight filler, and the secondcomponent may comprise about 95% by weight water, about 1.5% by weightsodium bicarbonate, and about 1% by weight of a source of peroxygen. Inother instances, the first component may comprise about 55.5% by weighttriacetin, about 41% by weight tripropylene glycol methyl ether, about0.9% sodium bicarbonate, about 0.3% by weight of a spray dried enzymepowder comprising Thermotoga neapolitana or Thermotoga maritimaperhydrolase or a variant derivative of Thermotoga neapolitana orThermotoga maritima perhydrolase having one or more point mutations thatimprove the perhydrolysis activity, and about 2.5% by weight fumedsilica, and the second component may comprise about 96% by weight water,about 0.2% by weight hydrogen peroxide stabilizer (e.g.,(1-hydroxyethylidene)bisphosphonic acid)), and about 3.2% by weight of asolution comprising 30% hydrogen peroxide.

With respect to the present methods and systems, the carboxylic acidester substrate may be used at a concentration sufficient to produce thedesired concentration of peracid upon enzyme-catalyzed perhydrolysis.The carboxylic acid ester need not be completely soluble in the reactionformulation, but preferably has sufficient solubility to permitconversion of the ester by the perhydrolase catalyst to thecorresponding peracid. The carboxylic acid ester is present in theaqueous reaction formulation at a concentration of about 0.0005 wt % toabout 40 wt % of the reaction formulation, preferably at a concentrationof 0.1 wt % to 20 wt % of the aqueous reaction formulation, and morepreferably at a concentration of 0.5 wt % to 10 wt % of the aqueousreaction Formulation. The wt % of carboxylic acid ester may be greaterthan the solubility limit of the carboxylic acid ester. Not all of theadded carboxylic acid ester must immediately dissolve in the aqueousreaction formulation, and after an initial mixing of all reactioncomponents, additional continuous or discontinuous mixing is optional.

In the present methods and systems, the peroxygen source may include,but is not limited to, hydrogen peroxide, hydrogen peroxide adducts(such as urea-hydrogen peroxide adduct (carbamide peroxide)), perboratesalts and percarbonate salts. The concentration of peroxygen compound inthe aqueous reaction formulation may range from 0.0033 wt % to about 50wt %, preferably from 0.033 wt % to about 40wt %, more preferably from0.33 wt % to about 30 wt %.

The reaction formulation of the present methods and systems may comprisefrom about 5 mM to about 250 mM substrate, from about 5 mM to about 250mM of the source of peroxygen, and from about 0.0001 mg/mL to about 10mg/mL, preferably about 0.01 mg/mL to about 2.0 mg/mL, of the enzymecatalyst. In such instances, the substrate may comprise triacetin, thesource of peroxygen may comprise hydrogen peroxide, and enzyme catalystmay comprise Thermotoga neapolitana or Thermotoga maritima perhydrolase.In another embodiment, the enzyme catalyst may comprise a Thermotoganeapolitana or Thermotoga maritima variant as defined by SEQ ID NO: 73or SEQ ID NO: 74, where the wild type cysteine at amino acid residueposition 277 is substituted with alanine, valine, serine, or threonine.

Many perhydrolase catalysts (such as whole cells, permeabilized wholecells, and partially purified whole cell extracts) have been reported tohave catalase activity (EC 1.11.1.6). Catalases catalyze the conversionof hydrogen peroxide into oxygen and water. In one aspect of thepresently disclosed methods and systems, the perhydrolysis catalystlacks catalase activity. In another aspect, a catalase inhibitor isadded to the reaction formulation. Examples of catalase inhibitorsinclude, but are not limited to, sodium azide and hydroxylamine sulfate.One of skill in the art can adjust the concentration of catalaseinhibitor as needed. The concentration of the catalase inhibitortypically ranges from 0.1 mM to about 1 M; preferably about 1 mM toabout 50 mM; more preferably from about 1 mM to about 20 mM. In oneaspect, sodium azide concentration typically ranges from about 20 mM toabout 60 mM while hydroxylamine sulfate is concentration is typicallyabout 0.5 mM to about 30 mM, preferably about 10 mM.

In other embodiments of the present methods and systems, the enzymecatalyst lacks significant catalase activity or is engineered todecrease or eliminate catalase activity. The catalase activity in a hostcell can be down-regulated or eliminated by disrupting expression of thegene(s) responsible for the catalase activity using well knowntechniques including, but not limited to, transposon mutagenesis, RNAantisense expression, targeted mutagenesis, and random mutagenesis. Insome embodiments of the present methods and systems, the gene(s)encoding the endogenous catalase activity are down-regulated ordisrupted (i.e., a “knocked-out”). As used herein, a “disrupted” gene isone where the activity and/or function of the protein encoded by themodified gene is no longer present. Means to disrupt a gene arewell-known in the art and may include, but are not limited to,insertions, deletions, or mutations to the gene so long as the activityand/or function of the corresponding protein is no longer present. In afurther preferred embodiment, the production host is an E. coliproduction host comprising a disrupted catalase gene selected from thegroup consisting of katG (SEQ ID NO: 35) and katE (SEQ ID NO: 44). Inother embodiments, the production host is an E. coli strain comprising adown-regulation and/or disruption in both katg1 and katE catalase genes.An E. coli strain comprising a double-knockout of katG and katE isprovided herein (E. coli strain KLP18).

The catalase negative E. coli strain KLP18 described herein has beendemonstrated to be a superior host for large scale (10-L and greater)production of perhydrolase enzymes compared to the catalase negativestrain UM2 (E. coli Genetic Stock Center #7156, Yale University, NewHaven Conn.), as determined by growth under fermenter conditions.Although both KLP18 and UM2 are catalase-negative strains, UM2 is knownto have numerous nutritional auxotrophies, and therefore requires mediathat is enriched with yeast extract and peptone. Even when employingenriched media for fermentation, UM2 grew poorly and to a limitedmaximum cell density (OD). In contrast, KLP18 had no special nutritionalrequirements and grew to high cell densities on mineral media alone orwith additional yeast extract.

The concentration of the catalyst in the aqueous reaction formulation ofthe present methods and systems depends on the specific catalyticactivity of the catalyst and is chosen to obtain the desired rate ofreaction. The weight of catalyst in perhydrolysis reactions typicallyranges from 0.0001 mg to 10 mg per mL of total reaction volume,preferably from 0.010 mg to 2.0 mg per mL. The catalyst may also beimmobilized on a soluble or insoluble support using methods well-knownto those skilled in the art; see for example, Immobilization of Enzymesand Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J.,USA; 1997. The use of immobilized catalysts permits the recovery andreuse of the catalyst in subsequent reactions. The enzyme catalyst maybe in the form of whole microbial cells, permeabilized microbial cells,microbial cell extracts, partially-purified or purified enzymes, ormixtures thereof.

In one aspect of the present methods and systems, the concentration ofperacid generated by the combination of chemical perhydrolysis andenzymatic perhydrolysis of the carboxylic acid ester is sufficient toprovide an effective concentration of peracid for bleaching,sanitization or disinfection at a desired pH. In another aspect, thepresent methods and systems provide combinations of enzymes and enzymesubstrates to produce the desired effective concentration of peracid,where, in the absence of added enzyme, there is a significantly lowerconcentration of peracid produced. Although there may in some cases besubstantial chemical perhydrolysis of the enzyme substrate by directchemical reaction of inorganic peroxide with the enzyme substrate, theremay not be a sufficient concentration of peracid generated to provide aneffective concentration of peracid in the desired applications, and asignificant increase in total peracid concentration is achieved by theaddition of an appropriate perhydrolase catalyst to the reactionformulation.

In connection with the present systems and methods, the concentration ofperacid generated (e.g., peracetic acid) by the perhydrolysis of atleast one carboxylic acid ester may be at least about 2 ppm, preferablyat least 20 ppm, preferably at least 100 ppm, more preferably at leastabout 200 ppm peracid, more preferably at least 300 ppm, more preferablyat least 500 ppm, more preferably at least 700 ppm, more preferably atleast about 1000 ppm peracid, most preferably at least 2000 ppm peracidwithin 10 minutes, preferably within 5 minutes, and most preferablywithin 1 minute of initiating the perhydrolysis reaction. The productformulation comprising the peracid may be optionally diluted with water,or a solution predominantly comprised of water, to produce a formulationwith the desired lower concentration of peracid. In one aspect of thepresent methods and systems, the reaction time required to produce thedesired concentration of peracid is not greater than about two hours,preferably not greater than about 30 minutes, more preferably notgreater than about 10 minutes, even more preferably not greater thanabout 5 minutes, and most preferably in about 1 minute or less. In otheraspects of the present methods for disinfecting a surface, a surface,including a hard surface or inanimate object, contaminated with abiological contaminant(s) is contacted with the peracid formed inaccordance with the processes described herein within about 1 minute toabout 168 hours of combining said reaction components, or within about 1minute to about 48 hours, or within about 1 minute to 2 hours ofcombining said reaction components, or any such time interval therein,

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of thereaction formulation (approximately 0° C.) to about 95° C., preferablyabout 5° C. to about 75° C., with a more preferred range of reactiontemperature of from about 5° C. to about 55° C.

The pH of the final reaction formulation containing peracid is fromabout 2 to about 9, preferably from about 3 to about 8, more preferablyfrom about 5 to about 8, even more preferably about 6 to about 8, andyet even more preferably about 6.5 to about 7.5. In another embodiment,the pH of the reaction formulation is acidic (pH<7). The pH of thereaction, and of the final reaction formulation, may optionally becontrolled by the addition of a suitable buffer, including, but notlimited to, bicarbonate, citrate, acetate, phosphate, pyrophosphate,methylphosphonate, succinate, malate, fumarate, tartrate, or maleate.The concentration of buffer, when employed, is typically from 0.1 mM to1.0 M, preferably from 1 mM to 300 mM, most preferably from 10 mM to 100mM.

In another aspect of the present methods and systems, the enzymaticperhydrolysis product may contain additional components that providedesirable functionality. In a further aspect, the present methods andsystems are used in laundry care applications for the treatment of anarticle of clothing. Examples of additional components include, but arenot limited to, buffers, detergent builders, thickening agents,emulsifiers, surfactants, wetting agents, corrosion inhibitors (such asbenzotriazole), enzyme stabilizers, and hydrogen peroxide stabilizers(such as metal ion chelating agents). Many of the additional componentsare well known in the detergent industry (such as those described inU.S. Pat. No. 5,932,532; hereby incorporated by reference). Examples ofemulsifiers include, but are not limited to, polyvinyl alcohol orpolyvinylpyrrolidone. Examples of thickening agents include, but are notlimited to, LAPONITE® RD, corn starch, PVP, CARBOWAX®, CARBOPOL®,CABOSIL®, polysorbate 20, PVA, and lecithin. Examples of bufferingsystems include, but are not limited to, sodium phosphatemonobasic/sodium phosphate dibasic; sulfamic acid/triethanolamine;citric acid/triethanolamine; tartaric acid/triethanolamine; succinicacid/triethanolamine; and acetic acid/triethanolamine. Examples ofsurfactants include, but are not limited to, (a) non-ionic surfactantssuch as block copolymers of ethylene oxide or propylene oxide,ethoxylated or propoxylated linear and branched primary and secondaryalcohols, and aliphatic phosphine oxides; (b) cationic surfactants suchas quaternary ammonium compounds, particularly quaternary ammoniumcompounds having a C8-C20 alkyl group bound to a nitrogen atomadditionally bound to three C1-C2 alkyl groups; (c) anionic surfactantssuch as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkylphosphonates, alkane sulfonates (e.g., sodium dodecylsulphate “SDS”) orlinear or branched alkyl benzene sulfonates, alkene sulfonates; and (d)amphoteric and zwitterionic surfactants such as aminocarboxylic acids,aminodicarboxylic acids, alkybetaines, and mixtures thereof. Additionalcomponents may include fragrances, dyes, stabilizers of hydrogenperoxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc.,St. Louis, Mo. and ethylenediaminetetraacetic acid (EDTA)), TURPINAL® SL(CAS#2809-21-4), DEQUEST® 0520, DEQUEST® 0531, stabilizers of enzymeactivity (e.g., polyethylene glycol (PEG)), and detergent builders.

In another aspect of the present methods of disinfecting a surface, theenzymatic perhydrolysis product may be pre-mixed to generate the desiredconcentration of peroxycarboxylic acid prior to contacting the surfaceto be disinfected. In another aspect of the present methods ofdisinfecting a surface, the enzymatic perhydrolysis product is notpre-mixed to generate the desired concentration of peroxycarboxylic acidprior to contacting the surface (such as a hard surface or inanimateobject) to be disinfected, but instead, the components of the reactionformulation that generate the desired concentration of percarboxylicacid are contacted with the surface to be disinfected, sanitized,bleached, destained, deodorized or any combination thereof, generatingthe desired concentration of peroxycarboxylic acid. In some embodiments,the components of the reaction formulation combine or mix at the locus.In some embodiments, the reaction components are delivered or applied tothe locus and subsequently mix or combine to generate the desiredconcentration of peroxycarboxylic acid.

In any embodiment of the present methods of disinfecting a surface ordelivering a benefit to an article of clothing or textile (stainremoval, order reduction, bleaching, sanitization, and/or disinfection),the aqueous reaction formulation may be applied to the surface byspraying, pouring, sprinkling, wiping, or by any other suitabletechnique, of which numerous other examples will be appreciated amongthose skilled in the art.

In Situ Production of Peracids Using a Perhydrolase Catalyst

Cephalosporin C deacetylases (EC. 3.1.1.41; systematic namecephalosporin C acetylhydrolases; CAHs) are enzymes having the abilityto hydrolyze the acetyl ester bond on cephalosporins such ascephalosporin C, 7-aminocephalosporanic acid, and7-(thiophene-2-acetamido)cephalosporanic acid (Abbott, B. and Fukuda,D., Appl. Microbiol. 30(3):413-419 (1975)). CAHs belong to a largerfamily of structurally related enzymes referred to as the carbohydrateesterase family seven (CE-7; see Coutinho, P. M., Henrissat, B.“Carbohydrate-active enzymes: an integrated database approach” in RecentAdvances in Carbohydrate Bioengineering, H. J. Gilbert, G. Davies, B.Henrissat and B. Svensson eds., (1999) The Royal Society of Chemistry,Cambridge, pp. 3-12.)

The CE-7 family includes both CAHs and acetyl xylan esterases (AXEs;E.C. 3.1.1.72). CE-7 family members share a common structural motif andare quite unusual in that they typically exhibit ester hydrolysisactivity for both acetylated xylooligosaccharides and cephalosporin C,suggesting that the CE-7 family represents a single class of proteinswith a multifunctional deacetylase activity against a range of smallsubstrates (Vincent et al., supra). Vincent et al. describes thestructural similarity among the members of this family and defines asignature sequence motif characteristic of the CE-7 family (“the CE-7signature motif”).

Members of the CE-7 family are found in plants, fungi (e.g.,Cephalosporidium acremonium), yeasts (e.g., Rhodosporidium toruloides,Rhodotorula glutinis), and bacteria such as Thermoanaerobacterium sp.;Norcardia lactamdurans, and various members of the genus Bacillus(Politino et al., Appl. Environ. Microbiol., 63(12):4807-4811 (1997);Sakai et al., J. Ferment Bioeng. 85:53-57 (1998); Lorenz, W. and Wiegel,J., J. Bacterial 179:5436-5441 (1997); Cardoza at al., Appl. Microbiol.Biotechnol., 54(3):406-412 (2000); Mitsushima et al., supra, Abbott, B.and Fukuda, D., Appl. Microbial. 30(3):413-419 (1975); Vincent et al.,supra, Takami at al., NAR, 28(21):4317-4331 (2000); Rey at al., GenomeBiol., 5(10): article 77 (2004); Degrassi et al., Microbiology.,146:1585-1591 (2000); U.S. Pat. No. 6,645,233; U.S. Pat. No. 5,281,525;U.S. Pat. No. 5,338,676; and WO 99/03984. A non-comprehensive list ofCE-7 carbohydrate esterase family members having significant homology toSEQ ID NO: 2 are provided in Table 1.

TABLE 1 Examples of CE-7 Enzymes Having Significant Homology to SEQ IDNO: 2. % Amino Source Organism Acid (GENBANK ® Nucleotide Amino AcidIdentity to Accession No. of Sequence Sequence SEQ ID the CE-7 enzyme)(SEQ ID NO:) (SEQ ID NO:) NO: 2. Reference B. subtilis  1 2 100 B.subtilis ATCC ® 31954 ™ SHS 0133 Mitsushima et al., supra B. subtilissubsp.  3 4 98 Kunst et al., subtilis str. 168 supra. (NP_388200)WO99/03984 B. subtilis BE1010 Payne and Jackson, J. Bacteriol. 173:2278-2282 (1991)) B. subtilis  5 6 96 U.S. Pat. No. ATCC ® 66336,465,233 (YP_077621.1) B. subtilis 25 26 96 Abbott and ATCC ® 29233 ™Fukuda, supra B. licheniformis  7 8 77 Rey et al., supra ATCC ® 14580 ™(YP_077621.1) B. pumilus PS213 9, 48 10 76 Degrassi et al., (CAB76451.2)supra Clostridium 11 12 57 Copeland et al. thermocellum US Dept. ofATCC ® 27405 ™ Energy Joint (ZP_00504991) Genome Institute (JGI- PGF)Direct Submission GENBANK ® ZP_00504991 Thermotoga 13, 29 14 42 Seeneapolitana GENBANK ® (AAB70869.1) AAB70869.1 Thermotoga 15, 56 16 42Nelson et al., maritima MSB8 Nature 399 (NP_227893.1) (6734): 323-329(1999) Bacillus sp. 19 20 40 Siefert et al. NRRL B-14911 J. Craig Venter(ZP_01168674) Institute. Direct Submission Under GENBANK ® ZP_01168674Thermoanaerobacterium 17 18 37 Lorenz and sp. Wiegel, supra (AAB68821.1)Bacillus 21 22 36 Takami et al., halodurans C-125 supra (NP_244192)Thermoanearobacterium 53 54 35 Lee, Y. E. and saccharolyticum Zeikus, J.G., J (S41858) Gen Microbiol. (1993), 139 Pt 6: 1235-1243 Bacillusclausii 23, 51 24 33 Kobayashi et KSM-K16 al., Appl. (YP_175265)Microbiol. Biotechnol. 43 (3), 473-481 (1995) Thermotoga 57, 58, and 5960 37 Copeland et al. lettingae US Dept. of (CP000812) Energy JointGenome Institute Direct Submission GENBANK ® CP000812 Thermotoga 61, 62,and 63 64 41 Copeland et al. Petrophila US Dept. of (CP000702) EnergyJoint Genome Institute Direct Submission GENBANK ® CP000702 Thermotogasp. 65, 66, and 67 68 42 Copeland et al. RQ2 US Dept. of “RQ2(a)” EnergyJoint (CP000969) Genome Institute Direct Submission GENBANK ® CP000969Thermotoga sp. 69, 70, and 71 72 42 Copeland et al. RQ2 US Dept. of“RQ2(b)” Energy Joint (CP000969) Genome Institute Direct SubmissionGENBANK ® CP000969

The perhydrolases for use in the present methods and systems arepreferably all members of the CE-7 carbohydrate esterase family. Theenzyme catalyst may comprise Thermotoga neapolitana perhydrolase definedby SEQ ID NO:14. In other embodiments of the present methods andsystems, the enzyme catalyst may comprise Thermotoga maritimaperhydrolase defined by SEQ ID NO:16. As described by Vincent et al.(supra), members of the family share a common signature motif that ischaracteristic of this family. A CLUSTALW alignment of the presentperhydrolases illustrates that all of the members belong to the CE-7carbohydrate esterase family (FIG. 1, sheets A-F). A comparison of theoverall percent amino acid identity among several of CE-7 perhydrolasesis provided in Table 2,

TABLE 2 Percent Amino Acid Identity Between Perhydrolases¹ 1 2 3 4 5 6 78 9 10 11 12 13 14 15 1 100 2 99 100 3 99 99 100 4 96 96 97 100 5 77 7677 76 100 6 76 76 76 76 68 100 7 57 57 57 56 56 56 100 8 42 43 43 43 4342 41 100 9 42 43 42 43 43 42 42 72 100 10 42 43 43 43 44 42 43 71 91100 11 41 43 43 43 45 42 43 71 97 91 100 12 41 42 42 42 43 41 42 71 9891 97 100 13 37 37 37 36 39 38 38 64 65 67 66 65 100 14 34 36 35 36 3536 33 36 32 34 34 33 36 100 15 33 34 33 33 32 34 32 30 30 32 31 31 32 34100 ¹= Percent Identity determined using blast2seq algorithm usingBLOSUM62, gap open = 11, gap extension = 1, x_drop = 0, expect = 10, andwordsize = 3. Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequence”,FEMS Microbial Lett. 174: 247-250

1. B. subtilis ATCC® 31954™

2. B. subtilis BE1010

3. B. subtilis ATCC® 29233™

4. B. subtilis ATCC® 6633™

5. B. licheniformis 14580

6. B. pumilus PS213

7. C. thermocellum ATCC® 27405™

8. Thermotoga sp. RQ2(b)

9. Thermotoga sp. RQ2(a)

10. T. neapolitana

11. T. maritima

12. T. petrophila

13. T. lettingae

14. T. saccharolyticum

15. B. clausii

Although variation is observed in terms of overall percent amino acididentity (i.e., the Clostridium thermocellum ATCC® 27405™ perhydrolase;SEQ ID NO: 12 shares only 57% amino acid identity with the Bacillussubtilis ATCC® 31954™ perhydrolase; SEQ ID NO: 2, while the Bacillusclausii perhydrolase (SEQ ID NO: 24) shares only 33% identity with SEQID NO: 2), each of the present perhydrolase enzymes share the CE-7signature motif. Accordingly, the perhydrolase catalyst of the presentinvention is an enzyme structurally classified as belonging to the CE-7carbohydrate esterase family. Each of the present perhydrolase enzymescomprises the CE-7 signature motif.

In one embodiment of the present methods and systems, suitableperhydrolytic enzymes can be identified by the presence of the CE-7signature motif (Vincent et al., supra). In preferred embodiments,perhydrolases comprising the CE-7 signature motif are identified using aCLUSTALW alignment against the Bacillus subtilis ATCC® 31954™perhydrolase (SEQ ID NO: 2; the reference sequence used for relativeamino acid position numbering). As per the amino acid residue numberingof SEQ ID NO: 2, the CE-7 signature motif comprises 3 conserved motifsdefined as: a) Arg118-Gly119-Gln120; b)Gly179-Xaa180-Ser181-Gln182-Gly183; and c) His298-Glu299. Thus, theenzyme catalyst used in the present systems and methods may be comprisethree sequence motifs defined as Arg118-Gly119-Gln120;Gly179-Xaa180-Ser181-Gln182-Gly183; and His298-Glu299 relative to SEQ IDNO:2. Alignments of the respective signature motifs are provided inTable 3. Typically, the Xaa at amino acid residue position 180 isglycine, alanine, proline, tryptophan, or threonine. Two of the threeamino acid residues belonging to the catalytic triad are in bold. In oneembodiment, the Xaa at amino acid residue position 180 is selected fromthe group consisting of glycine, alanine, proline, tryptophan, andthreonine.

Further analysis of the conserved motifs within the GE-7 carbohydrateesterase family indicates the presence of an additional motif (LXD atamino acid positions 267-269 of SEQ ID NO: 2) that may be used tofurther define a perhydrolase belonging to the CE-7 carbohydrateesterase family. In a further embodiment of the present methods andsystems, the signature motif defined above may include a fourthconserved motif defined as Leu267-Xaa268-Asp269 relative to referenceSEQ ID NO: 2. The Xaa at amino acid residue position 268 is typicallyisoleucine, valine, or methionine. The fourth motif includes theaspartic acid residue that is the third member of the catalytic triad(Ser181-Asp269-His298).

Any number of well-known global alignment algorithms may be used toalign two or more amino acid sequences (representing enzymes havingperhydrolase activity) to determine the existence of the presentsignature motif (for example, CLUSTALW or Needleman and Wunsch (J. Mol.Biol., 48:443-453 (1970)). The aligned sequence(s) is compared to thereference sequence (SEQ ID NO: 2). In one embodiment, a CLUSTALalignment (e.g., CLUSTALW; for example version 1.83)) using a referenceamino acid sequence (as used herein the CAH sequence (SEQ ID NO: 2) fromthe Bacillus subtilis ATCC® 31954™) is used to identify perhydrolasesbelonging to the CE-7 esterase family. The relative numbering of theconserved amino acid residues is based on the residue numbering of thereference amino acid sequence to account for small insertions ordeletions (typically 5 amino acids or less) within the aligned sequence.

A comparison of the overall percent identity among perhydrolasesexemplified herein indicates that enzymes having as little as 33%identity to SEQ ID NO: 2 (while retaining the signature motif) exhibitsignificant perhydrolase activity and are structurally classified asCE-7 carbohydrate esterases. In some embodiments of the present methodsand systems, the present perhydrolases include enzymes comprising thepresent signature motif and at least 30%, preferably at least 33%, morepreferably at least 40%, even more preferably at least 42%, even morepreferably at least 50%, even more preferably at least 60%, even morepreferably at least 70%, even more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID ND:2.

Examples of CE-7 enzymes comprised of the above signature motif areprovided in Table 3.

TABLE 3 Conserved motifs from several CE-7 carbohydrate esterases. GXSQGHE motif^(a) Perhydrolase RGQ motif^(a) motif^(a) LXD motif^(b) (ResidueSequence (Residue #s) (Residue #s) (Residue #s) #s) SEQ ID NO: 2 118-120179-183 267-269 298-299 SEQ ID NO: 4 118-120 179-183 267-269 298-299 SEQID NO: 6 118-120 179-183 267-269 298-299 SEQ ID NO: 8 119-121 180-184268-270 299-300 SEQ ID NO: 10 118-120 179-183 267-269 298-299 SEQ ID NO:12 119-121 181-185 269-271 300-301 SEQ ID NO: 14 118-120 186-190 272-274303-304 SEQ ID NO: 16 118-120 186-190 272-274 303-304 SEQ ID NO: 18117-119 180-184 270-272 301-302 SEQ ID NO: 20 118-120 178-182 267-269304-305 SEQ ID NO: 22 118-119 181-185 271-273 302-303 SEQ ID NO: 24117-119 180-184 270-272 301-302 SEQ ID NO: 26 118-120 179-183 267-269298-299 SEQ ID NO: 54 117-119 180-184 270-272 301-302 SEQ ID NO: 60118-120 186-190 272-274 303-304 SEQ ID NO: 64 118-120 186-190 272-274303-304 SEQ ID NO: 68 118-120 186-190 272-274 303-304 RQ2(a) SEQ ID NO:72 119-121 187-191 273-275 304-305 RQ2(b) SEQ ID NO: 73 118-120 186-190272-274 303-304 SEQ ID NO: 74 118-120 186-190 272-274 303-304 ^(a)=Conserved motifs defined by Vincent et al., supra used to define thesignature motif. ^(b)= an additional motif that may be useful in furtherdefining the signature motif defined by Vincent et al., supra.

Alternatively, a contiguous signature motif (SEQ ID NO: 49) comprisingthe 3 conserved motifs identified by Vincent of at, supra, (RGQ, GXSQG,and HE; Amino acids residues 118-299 of SEQ ID NO: 2; an optional 4^(th)motif, LXD, is also underlined) may also be used as a contiguoussignature motif to identify CE-7 carbohydrate esterases (FIG. 1, panelsA-F). As such, suitable enzymes expected to have perhydrolase activitymay also be identified as having at least 30% amino acid identify,preferably at least 36%, more preferably at least 40%, even morepreferably at least 50%, yet more preferably at least 60%, yet even morepreferably at least 70%, yet even more preferably at least 80%, yet evenmore preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% amino acid identity to SEQ ID NO: 49 (the 4 conserved motifs foundin CE-7 carbohydrate esterases are underlined).

(SEQ ID NO: 49) RGQQSSEDTSISLHGHALGWMTKGILDKDTYYYRGVYLDAVRALEVISSFDEVDETRIGVTGGSQGGGLTIAAAALSDIPKAAVADYPYLSNFERAIDVALEQPYLEINSFFRRNGSPETEVQAMKTLSYFDIMNLADRVKVPVLMSIGLIDKVTPPSTVFAAYNHLETEKELKVYRYFGHE.

A comparison using the contiguous signature sequence against severalCE-7 esterases having perhydrolase activity is provided in Table 4.BLASTP using default parameters was used.

TABLE 4 Percent Amino Acid Identity of Various CE-7 CarbohydrateEsterases having Perhydrolysis Activity Versus the Contiguous SignatureSequence (SEQ ID NO: 49). Perhydrolase % Identity using E-score SequenceBLASTP (expected) SEQ ID NO: 2 100 3e−92 SEQ ID NO: 4 98 6e−91 SEQ IDNO: 6 98 4e−98 SEQ ID NO: 8 78 1e−78 SEQ ID NO: 10 80 3e−76 SEQ ID NO:12 63 2e−56 SEQ ID NO: 14 51 1e−41 SEQ ID NO: 16 50 6e−35 SEQ ID NO: 2436 7e−21 SEQ ID NO: 26 99 2e−90 SEQ ID NO: 54 40 2e−26 SEQ ID NO: 60 403e−30 SEQ ID NO: 64 46 6e−35 SEQ ID NO: 68 46 6e−35 SEQ ID NO: 72 489e−36

Alternatively, the percent amino acid identity to the complete length ofone or more of the perhydrolases for use in the present methods andsystems may also be used. Accordingly, suitable enzymes havingperhydrolase activity have at least 30%, preferably at least 33%,preferably at least 40%, preferably at least 40%, more preferably atleast 50%, more preferably at least 60%, more preferably at least 70%,even more preferably at least 80%, yet even more preferably at least90%, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% amino acid identity to SEQ ID NO: 2. In furtherembodiments of the present methods and systems, suitable perhydrolasecatalysts comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO:54, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ IDNO: 73, and SEQ ID NO: 74. In another embodiment, the amino acid residueat position 277 of either SEQ ID NO: 73 or SEQ ID NO: 74 is selectedfrom the group consisting of alanine, valine, serine, and threonine. Inpreferred embodiments, suitable enzymes having perhydrolase activityhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% amino acid identity to SEQ ID NO: 14 or to SEQ ID NO: 16 may beused. In further preferred embodiments, suitable enzymes havingperhydrolase activity have an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 73, and SEQID NO: 74.

Suitable carbohydrate esterase family 7 (CE-7) enzymes havingperhydrolysis activity for use in the present methods and systems mayalso include enzymes having one or more deletions, substitutions, and/orinsertions to one of the present perhydrolase enzymes (such as SEQ IDNOs, 14 or 16). As shown in Table 2, CE-7 carbohydrates esterases havingperhydrolase activity share as little as 31% overall amino acididentity. Additional enzymes having perhydrolase activity structurallyclassified as belonging to the CE-7 carbohydrate esterase family mayhave even lower percent identity, so long as the enzyme retains theconserved signature motif. As such, the numbers of deletions,substitutions, and/or insertions may vary so long as the conservedsignature motifs (see Table 3) are found in their relative positionswithin the enzyme.

Additionally, it is well within one of skill in the art to identitysuitable enzymes according to the structural similarity found within thecorresponding nucleic acid sequence. Hybridization techniques can beused to identity similar gene sequences. Accordingly, suitableperhydrolase catalysts of the present invention comprise an amino acidsequence encoded by a nucleic acid molecule that hybridizes under highlystringent conditions to a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, SEQ ID NO:5; SEQ ID NO: 7; SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO: 25; SEQ ID NO:29; SEQ ID NO:48, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71.

Several variant CE-7 enzymes have recently been identified havingenhanced perhydrolysis activity relative to the corresponding wild-typeenzymes from which they were derived (see co-owned, co-filed, andcopending U.S. patent application having attorney docket number CL4392US NA; herein incorporated by reference). Specifically, severalwild-type CE-7 enzymes from the genus Thermotoga were modified toproduce several variants having enhanced perhydrolysis activity.Sequences of the variants are provided as SEQ ID NOs: 73 and 74 whereinthe amino acid residue at position 277 of either SEQ ID NO: 73 or SEQ IDNO: 74 is selected from the group consisting of alanine, valine, serine,and threonine.

Enzymatic Multi-Component Peracid Generation Systems

The present methods and systems may be used in the production ofindustrially useful, efficacious concentrations of peracids in situunder aqueous reaction conditions using the perhydrolase activity of anenzyme belonging to carbohydrate esterase family 7. In some embodiments,the enzyme having perhydrolase activity is also classified structurallyand functionally as a cephalosporin C deacetylase (CAH). In otherembodiments, the enzyme having perhydrolase activity is classifiedstructurally and functionally as an acetyl xylan esterase (AXE).

The peracids produced in accordance with the present methods and systemsare quite reactive and may decrease in concentration over extendedperiods of time, depending on variables including, but are not limitedto, temperature and pH. As such, it may be desirable to keep the variousreaction components separated, especially for liquid formulations. Inone aspect, the hydrogen peroxide source is separate from either thesubstrate or the perhydrolase catalyst, preferably from both. This canbe accomplished using a variety of techniques including, but not limitedto, the use of multi-compartment chambered dispensers (such as U.S. Pat.No. 4,585,150) and at the time of use physically combining theperhydrolase catalyst with an inorganic peroxide and the presentsubstrates to initiate the aqueous enzymatic perhydrolysis reaction. Theperhydrolase catalyst may optionally be immobilized within the body ofreaction chamber or separated (such as filtered) from the reactionproduct comprising the peracid prior to contacting the surface and/orobject targeted for treatment. The perhydrolase catalyst may be in aliquid matrix or in a solid form (such as a powder or tablet) orembedded within a solid matrix that is subsequently mixed with thesubstrates to initiate the enzymatic perhydrolysis reaction. In afurther aspect, the perhydrotase catalyst may be contained within adissolvable or porous pouch that may be added to the aqueous substratematrix to initiate enzymatic perhydrolysis. In additional furtheraspects, a powder comprising the enzyme catalyst is suspended in thecarboxylic acid ester substrate (such as triacetin) and at time of useis mixed with a source of peroxygen in water. In some embodiments, atwo-compartment spray bottle such as a dual-liquid fixed ratio sprayer(Model DLS100, Take 5 Corp., Rogue River, Oreg.) or a dual-liquidvariable ratio sprayer (Model DLS200, Take; 5 Corp.) is utilized (U.S.Pat. No. 5,152,461 and U.S. Pat. No. 5,532,157; herein incorporated byreference). In another embodiment, a single bottle containing twoseparate compartments separated by a breakable seal is employed. In someembodiments, the ratio of the volume of the two separate compartments is1:1, or 5:1 or 10:1. Examples of multi-component delivery systems mayalso be found in co-owned, co-filed, and copending U.S. patentapplication Attorney Docket No. CL4400 US NA (incorporated herein byreference in its entirety).

The presently disclosed systems for producing peroxycarboxylic acid maycomprise a first component comprising an enzyme catalyst (such as anenzyme powder), a carboxylic acid ester substrate (substantially free ofwater), and the cosolvent, and a second component comprising the sourceof peroxygen and water. The presence of excess water in the firstcomponent may lead to storage instability. As such, the phrase“substantially free of water” will refer to a concentration of water inthe component comprising the carboxylic acid estersubstrate/enzyme/cosolvent that does not adversely impact the storagestability of enzyme when present in the first component. In oneembodiment, “substantially free of water” may mean less than 2000 ppm,preferably less than 1000 ppm, more preferably less than 500 ppm, andeven more preferably less than 250 ppm of water in the carboxylic acidester substrate containing component. In such embodiments, the systemsmay further comprise a first vessel/container for storing the firstcomponent and a second vessel/container for storing the secondcomponent. As used herein, the term “container” may be used genericallyto describe vessels, compartments, bottles, packets, and other packingsystems suitable for holding and/or transporting the present materials.The present systems may also comprise a mixing compartment for receivingat least some of the first component from the first container and atleast some of the second component from the second container, therebypermitting the formation of a formulation comprising at least some ofthe first component and at least some of the second component. In suchinstances, the systems may further comprise a nozzle for dispensing theformulation from the mixing compartment.

In other embodiments of the present systems, the system comprising afirst and second vessel may further comprise a nozzle for receiving atleast some of the first component from the first vessel and at leastsome of the second component from the second vessel, and for dispensingat least some of the first component contemporaneously with at leastsome of the second component. As used herein, “contemporaneously” meansthat during at least part of the time that at least some of the firstcomponent is dispensed, at least some of the second component is alsodispensed. Thus, where some of the first component is dispensed for atotal duration of one second, dispensing the second component for 1second after dispensing the first component and for 0.1 seconds duringthe dispensing of the first component will be considered to have beencontemporaneous with the dispensing of the first component.

Methods for Determining the Concentration of Peracid and HydrogenPeroxide.

A variety of analytical methods may be used in the present methods toanalyze the reactants and products including, but not limited to,titration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the analytical procedure described by U. Karst et al., (Anal.Chem., 69(17) 3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S.Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO2004/058961 A1) as described in the present examples.

Determination of Minimum Biocidal Concentration of Peracids

The method described by Gabrielson, of al. (J. Microbiol. Methods 50:63-73 (2002)) may be employed for determination of the Minimum BiocidalConcentration (MBC) of peracids, or of hydrogen peroxide and enzymesubstrates. The assay method is based on XTT reduction inhibition, whereXTT((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to, viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically Prepared Peracid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced accordingto the present method can be used in a variety of hard surface/inanimateobject applications for reduction of concentrations of biologicalcontaminants, such as decontamination of medical instruments (e.g.,endoscopes), textiles (e.g., garments, carpets), food preparationsurfaces, food storage and food-packaging equipment, materials used forthe packaging of food products, chicken hatcheries and grow-outfacilities, animal enclosures, and spent process waters that havemicrobial and/or virucidal activity. The enzyme-generatedperoxycarboxylic acids may be used in formulations designed toinactivate prions (e.g., certain proteases) to additionally providebiocidal activity. In a preferred aspect, the present peroxycarboxylicacid compositions are particularly useful as a disinfecting agent fornon-autoclavable medical instruments and food packaging equipment. Asthe peroxycarboxylic acid-containing formulation may be prepared usingGRAS or food-grade components (enzyme, enzyme substrate, hydrogenperoxide, and buffer), the enzyme-generated peroxycarboxylic acid mayalso be used for decontamination of animal carcasses, meat, fruits andvegetables, or for decontamination of prepared foods. Theenzyme-generated peroxycarboxylic acid may be incorporated into aproduct whose final form is a powder, liquid, gel, film, solid oraerosol. The enzyme-generated peroxycarboxylic acid may be diluted to aconcentration that still provides an efficacious decontamination.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can be used to disinfect surfaces and/or objectscontaminated (or suspected of being contaminated) with biologicalcontaminants by contacting the surface or object with the productsproduced by the present processes. As used herein, “contacting” refersto placing a disinfecting composition comprising an effectiveconcentration of peroxycarboxylic acid in contact with the surface orinanimate object suspected of contamination with a biologicalcontaminant for a period of time sufficient to clean and disinfect.Contacting includes spraying, treating, immersing, flushing, pouring onor in, mixing, combining, painting, coating, applying, affixing to andotherwise communicating a peroxycarboxylic acid solution or compositioncomprising an efficacious concentration of peroxycarboxylic acid, or asolution or composition that forms an efficacious concentration ofperoxycarboxylic acid, with the surface or inanimate object suspected ofbeing contaminated with a concentration of a biological contaminant. Thedisinfectant compositions may be combined with a cleaning composition toprovide both cleaning and disinfection. Alternatively, a cleaning agent(e.g., a surfactant or detergent) may be incorporated into theformulation to provide both cleaning and disinfection in a singlecomposition.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can also contain at least one additionalantimicrobial agent, combinations of prion-degrading proteases, avirucide, a sporicide, or a biocide. Combinations of these agents withthe peroxycarboxylic acid produced by the claimed processes can providefor increased and/or synergistic effects when used to clean anddisinfect surfaces and/or objects contaminated (or suspected of beingcontaminated) with biological contaminants. Suitable antimicrobialagents include carboxylic esters (e.g., p-hydroxy alkyl benzoates andalkyl cinnamates); sulfonic acids (e.g., dodecylbenzene sulfonic acid);iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite); organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof; phenolic derivatives (such as o-phenylphenol, o-benzyl-p-chlorophenol, tent-amyl phenol and C₁-C₆ alkylhydroxy benzoates); quaternary ammonium compounds (such asalkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof); and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peroxycarboxylic acids formed by the present processcan be used to reduce the concentration of viable biologicalcontaminants (such as a viable microbial population) when applied onand/or at a locus. As used herein, a “locus” comprises part or all of atarget surface suitable for disinfecting or bleaching. Target surfacesinclude all surfaces that can potentially be contaminated withbiological contaminants. Non-limiting examples include equipmentsurfaces found in the food or beverage industry (such as tanks,conveyors, floors, drains, coolers, freezers, equipment surfaces, walls,valves, belts, pipes, drains, joints, crevasses, combinations thereof,and the like); building surfaces (such as wails, floors and windows);non-food-industry related pipes and drains, including water treatmentfacilities, pools and spas, and fermentation tanks; hospital orveterinary surfaces (such as walls, floors, beds, equipment (such asendoscopes), clothing worn in hospital/veterinary or other healthcaresettings, including clothing, scrubs, shoes, and other hospital orveterinary surfaces); restaurant surfaces; bathroom surfaces; toilets;clothes and shoes; surfaces of barns or stables for livestock, such aspoultry, cattle, dairy cows, goats, horses and pigs; hatcheries forpoultry or for shrimp; and pharmaceutical or biopharmaceutical surfaces(e.g., pharmaceutical or biopharmaceutical manufacturing equipment,pharmaceutical or biopharmaceutical ingredients, pharmaceutical orbiopharmaceutical excipients). Additional hard surfaces also includefood products, such as beef, poultry, pork, vegetables, fruits, seafood,combinations thereof, and the like. The locus can also include waterabsorbent materials such as infected linens or other textiles. The locusalso includes harvested plants or plant products including seeds, corms,tubers, fruit, and vegetables, growing plants, and especially cropgrowing plants, including cereals, leaf vegetables and salad crops, rootvegetables, legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used toprovide a benefit to a textile including, but not limited to, bleaching,destaining, sanitization, disinfection, and deodorizing. Theperoxycarboxylic acids formed by the present process may be used in anynumber of laundry care products including, but not limited to, textilepre-wash treatments, laundry detergents, stain removers, bleachingcompositions, deodorizing compositions, and rinsing agents.

Recombinant Microbial Expression

With respect to the present methods and systems, the genes and geneproducts of the sequences described herein may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the genes andnucleic acid molecules are microbial hosts that can be found within thefungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to, bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Phaffia, Candida, Hansenula,Yarrowia, Kluyveromyces, Salmonella, Bacillus, Acinetobacter, Zymomonas,Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobaciflus,Methanobacterium, Klebsiella, and Myxococcus. In one embodiment,bacterial host strains include Kluyveromyces, Escherichia, Bacillus, andPseudomonas. In a preferred embodiment, the bacterial host cell isEscherichia coli.

Large-scale microbial growth and functional gene expression may use awide range of simple or complex carbohydrates, organic acids andalcohols or saturated hydrocarbons, such as methane or carbon dioxide inthe case of photosynthetic or chemoautotrophic hosts, the form andamount of nitrogen, phosphorous, sulfur, oxygen, carbon or any tracemicronutrient including small inorganic ions. The regulation of growthrate may be affected by the addition, or not, of specific regulatorymolecules to the culture and which are not typically considered nutrientor energy sources.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell and/or native to theproduction host, although such control regions need not be so derived.

Initiation control regions or promoters, which are useful to driveexpression of the present cephalosporin C deacetylase coding region inthe desired host cell are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving these genes is suitablefor the present invention including but not limited to CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, araB, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred host cell. In one embodiment, the inclusion of atermination control region is optional. In another embodiment, thechimeric gene includes a termination control region derived thepreferred host cell.

Industrial Production

A variety of culture methodologies may be applied to produce the presentperhydrolase catalysts in accordance with the present methods andsystems. For example, large-scale production of a specific gene productoverexpressed from a recombinant microbial host may be produced by bothbatch and continuous culture methodologies.

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process, the media is inoculated with thedesired organism or organisms and growth or metabolic activity may occurwithout adding anything further to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon source,and attempts are often made to control factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the fed-batch system.Fed-batch culture processes are also suitable in the present inventionand comprise a typical batch system except that the substrate is addedin increments as the culture progresses. Fed-batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedia. Measurement of the actual substrate concentration in fed-batchsystems is difficult and is estimated on the basis of the changes ofmeasurable factors such as pH, dissolved oxygen and the partial pressureof waste gases such as CO₂. Batch and fed-batch culturing methods arecommon and well known in the art and examples may be found in Thomas D.Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition, Sinauer Associates, Inc., Sunderland, Mass. (1989) andDeshpande, Mukund V., Appl. Biochem. Biotechnol., 38:227-234 (1992).

Commercial production of the desired perhydrolase catalysts may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions, and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited to,monosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, methane or methanol (for example,when the host cell is a methylotrophic microorganism). Similarly,various species of Candida will metabolize alanine or oleic acid (Sulteret al., Arch. Microbiol., 153:485-489 (1990)). Hence, it is contemplatedthat the source of carbon utilized in the present invention mayencompass a wide variety of carbon-containing substrates and will onlybe limited by the choice of organism,

Recovery of the desired perhydrolase catalysts from a batchfermentation, fed-batch fermentation, or continuous culture, may beaccomplished by any of the methods that are known to those skilled inthe art. For example, when the enzyme catalyst is producedintracellularly, the cell paste is separated from the culture medium bycentrifugation or membrane filtration, optionally washed with water oran aqueous buffer at a desired pH, then a suspension of the cell pastein an aqueous buffer at a desired pH is homogenized to produce a cellextract containing the desired enzyme catalyst. The cell extract mayoptionally be filtered through an appropriate filter aid such as celiteor silica to remove cell debris prior to a heat-treatment step toprecipitate undesired protein from the enzyme catalyst solution. Thesolution containing the desired enzyme catalyst may then be separatedfrom the precipitated cell debris and protein by membrane filtration orcentrifugation, and the resulting partially-purified enzyme catalystsolution concentrated by additional membrane filtration, then optionallymixed with an appropriate carrier (for example, maltodextrin, phosphatebuffer, citrate buffer, or mixtures thereof) and spray-dried to producea solid powder comprising the desired enzyme catalyst.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given either as a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope be limited to the specific values recited whendefining a range.

GENERAL METHODS

The following examples are provided to demonstrate preferred aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the present inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the present invention.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma/Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “μL”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “mg” means milligram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “g” means gravity, “HPLC” meanshigh performance liquid chromatography, “dd H₂O” means distilled anddeionized water, “dcw” means dry cell weight, “ATCC” or “ATCC®” meansthe American Type Culture Collection (Manassas, Va.), “U” means unit(s)of perhydrolase activity, “rpm” means revolution(s) per minute, and“EDTA” means ethylenediaminetetraacetic acid.

Example 1 Construction of a katG Catalase Disrupted E. coli Strain

The coding region of the kanamycin resistance gene (kan; SEQ ID NO: 30)was amplified from the plasmid pKD13 (SEQ ID NO: 31) by PCR (0.5 min at94° C., 0.5 min at 55° C., 1 min at 70° C., 30 cycles) using primersidentified as SEQ ID NO: 32 and SEQ ID NO: 33 to generate the PCRproduct identified as SEQ ID NO: 34. The katG nucleic acid sequence isprovided as SEQ ID NO: 35 and the corresponding amino acid sequence isSEQ ID NO: 36. E. coli MG1655 (ATCC® 47076™) was transformed with thetemperature-sensitive plasmid pKD46 (SEQ ID NO: 37), which contains theλ-Red recombinase genes (Datsenko and Wanner, 2000, PAPAS USA97:6640-6645), and selected on LB-amp plates for 24 h at 30° C.MG1655/pKD46 was transformed with 50-500 ng of the PCR product byelectroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25μF), and selected on LB-kan plates for 24 h at 37° C. Several colonieswere streaked onto LB-kan plates and incubated overnight at 42° C. tocure the pKD46 plasmid. Colonies were checked to confirm a phenotype ofkanR/ampS. Genomic DNA was isolated from several colonies using thePUREGENE® DNA purification system (Gentra Systems, Inc., Minneapolis,Minn.), and checked by PCR to confirm disruption of the katG gene usingprimers identified as SEQ ID NO: 38 and SEQ ID NO: 39. SeveralkatG-disrupted strains were transformed with the temperature-sensitiveplasmid pCP20 (SEQ ID NO: 40), which contains the FLP recombinase, usedto excise the kan gene, and selected on LB-amp plates for 24 h at 37°C., Several colonies were streaked onto LB plates and incubatedovernight at 42° C. to cure the pCP20 plasmid. Two colonies were checkedto confirm a phenotype of kanS/ampS, and called MG1655 KatG1 and MG1655KatG2.

Example 2 Construction of a katE Catalase Disrupted E. coli Strain

The kanamycin resistance gene (SEQ ID NO: 30) was amplified from theplasmid pKD13 (SEQ ID NO: 31) by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:41 and SEQ ID NO: 42 to generate the PCR product identified as SEQ IDNO: 43. The katE nucleic acid sequence is provided as SEQ ID NO: 44 andthe corresponding amino acid sequence is SEQ ID NO: 45. E. coli MG1655(ATCC® 47076™) was transformed with the temperature-sensitive plasmidpKD46 (SEQ ID NO: 37), which contains the λ-Red recombinase genes, andselected on LB-amp plates for 24 h at 30° C. MG1655/pKD46 wastransformed with 50-500 ng of the PCR product by electroporation (BioRadGene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF), and selected onLB-kan plates for 24 h at 37° C. Several colonies were streaked ontoLB-kan plates and incubated overnight at 42° C. to cure the pKD46plasmid. Colonies were checked to confirm a phenotype of kanR/ampS.Genomic DNA was isolated from several colonies using the PUREGENE® DNApurification system, and checked by PCR to confirm disruption of thekatE gene using primers identified as SEQ ID NO: 46 and SEQ ID NO: 47.Several katE-disrupted strains were transformed with thetemperature-sensitive plasmid pCP20 (SEQ ID NO: 40), which contains theFLP recombinase, used to excise the kan gene, and selected on LB-ampplates for 24 h at 37° C. Several colonies were streaked onto LB platesand incubated overnight at 42° C. to cure the pCP20 plasmid. Twocolonies were checked to confirm a phenotype of kanS/ampS, and namedMG1655 KatE1 and MG1655 KatE2.

Example 3 Construction of a katG Catalase and katE Catalase Disrupted E.coli Strain (KLP18)

The kanamycin resistance gene (SEQ ID NO: 30) was amplified from theplasmid pKD13 (SEQ ID NO: 31) by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:41 and SEQ ID NO: 42 to generate the PCR product identified as SEQ IDNO: 43. E. coli MG1655 KatG1 was transformed with thetemperature-sensitive plasmid pKD46 (SEQ ID NO: 37), which contains theλ-Red recombinase genes, and selected on LB-amp plates for 24 h at 30°C. MG1655 KatG1/pKD46 was transformed with 50-500 ng of the PCR productby electroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W,25 μF), and selected on LB-kan plates for 24 h at 37° C. Severalcolonies were streaked onto LB-kan plates and incubated overnight at 42°C. to cure the pKD46 plasmid. Colonies were checked to confirm aphenotype of kanR/ampS. Genomic DNA was isolated from several coloniesusing the PUREGENE® DNA purification system, and checked by PCR toconfirm disruption of the katE gene using primers identified as SEQ IDNO: 46 and SEQ ID NO: 47. Several katE-disrupted strains (A katE) weretransformed with the temperature-sensitive plasmid pCP20 (SEQ ID NO:40), which contains the FLP recombinase, used to excise the kan gene,and selected on LB-amp plates for 24 h at 37° C. Several colonies werestreaked onto LB plates and incubated overnight at 42° C. to cure thepCP20 plasmid. Two colonies were checked to confirm a phenotype ofkanS/ampS, and named MG1655 KatG1KatE18.1 and MG1655 KatG1KatE23. MG1655KatG1KatE18.1 is designated E. coli KLP18.

Example 4 Cloning and Expression of Perhydrolase from Thermotoganeapolitana

The coding region of the gene encoding acetyl xylan esterase fromThermotoga neapolitana as reported in GENBANK® (Accession No. AAB70869)was synthesized using codons optimized for expression in E, coil (DNA2.0, Menlo Park, Calif.), The coding region of the gene was subsequentlyamplified by PCR (0.5 min at 94° C., 0.5 min at 55° C., 1 min at 70° C.,30 cycles) using primers identified as SEQ ID NO: 27 and SEQ ID NO: 28.The resulting nucleic acid product (SEQ ID NO: 29) was subcloned intopTrcHis2-TOPO® to generate the plasmid identified as pSW196. The plasmidpSW196 was used to transform E. coli KLP18 to generate the strainKLP18/pSW196, KLP18/pSW196 was grown in LB media at 37° C. with shakingup to OD_(600nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h, Cells wereharvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase at 20-40% of total soluble protein.

Example 5 Fermentation of E. coli KLP18 Transformants ExpressingPerhydrolase

A fermentor seed culture was prepared by charging a 2-L shake flask with0.5 L seed medium containing yeast extract (Amberex 695, 5.0 g/L),K₂HPO₄ (10.0 g/L), KH₂PO₄ (7.0 g/L), sodium citrate dihydrate (1.0 g/L),(NH₄)₂SO₄ (40 g/L), MgSO₄ heptahydrate (1.0 g/L) and ferric ammoniumcitrate (0.10 g/L). The pH of the medium was adjusted to 6.3 and themedium was sterilized in the flask. Post sterilization additionsincluded glucose (50 wt %, 10.0 mL) and 1 mL ampicillin (25 mg/mL) stocksolution. The seed medium was inoculated with a 1-mL culture of E. coliKLP18/pSW196 in 20% glycerol, and cultivated at 35° C. and 300 rpm. Theseed culture was transferred at ca. 1-2 OD₅₅₀ to a 14 L fermenter(Braun) with 8 L of medium at 35° C. containing KH₂PO₄ (3.50 g/L), FeSO₄heptahydrate (0.05 g/L), MgSO₄ heptahydrate (2.0 g/L), sodium citratedihydrate (1.90 g/L), yeast extract (Amberex 695, 5.0 g/L),Biospumex153K antifoam (0.25 mL/L, Cognis Corporation), NaCl (1.0 g/L),CaCl₂ dihydrate (10 g/L), and NIT trace elements solution (10 mL/L). Thetrace elements solution contained citric acid monohydrate (10 g/L),MnSO₄ hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L), ZnSO₄heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization additions included glucosesolution (50% w/w, 80.0 g) and ampicillin (25 mg/mL) stock solution(16.00 mL). Glucose solution (50% w/w) was used for fed batch. Glucosefeed was initiated when glucose concentration decreased to 0.5 g/L,starting at 0.31 g feed/min and increasing progressively each hour to0.36, 0.42, 0.49, 0.57, 0.66, 0.77, 0.90, 1.04, 1.21, 1.41 1.63 g/minrespectively; the rate remained constant afterwards. Glucoseconcentration in the medium was monitored and if the concentrationexceeded 0.1 g/L the feed rate was decreased or stopped temporarily.Induction was initiated between OD₅₅₀=56 and OD₅₅₀=80 with addition of16 mL IPTG (0.5 M) for the various strains. The dissolved oxygen (DO)concentration was controlled at 25% of air saturation. The DO wascontrolled first by impeller agitation rate (400 to 1400 rpm) and laterby aeration rate (2 to 10 slpm). The pH was controlled at 6.8. NH₄OH(29% w/w) and H₂SO₄ (20% w/v) were used for pH control. The headpressure was 0.5 bars. The cells were harvested by centrifugation 16 hpost IPTG addition.

Example 6 Preparation of Spray-Dried Thermotoga neapolitana Perhydrolase

Thermotoga neapolitana cell paste prepared as described in Example 5 wassuspended at a final concentration of 200 g wet cell weight/L in 50 mMsodium phosphate buffer at pH 7.4. The cells in the suspension werelysed in a single pass through an APV1000 homogenizer operated at 12,000psi (˜82.74 MPa) inlet pressure. The resulting lysate was heat treatedat 65° C. for approximately 30 minutes, and the homogenate cooled toroom temperature, and the resulting solids removed by centrifugation.The supernatant from the centrifugation was filtered using a 0.1 micronfilter, and the resulting filtrate containing perhydrolase wasconcentrated using a 30K NMWCO filter to a final protein concentrationof 34 mg protein/mL. To the protein solution was added maltodextrin to aconcentration of ca. 3-fold by weight that of the concentration ofprotein, and the resulting solution spray-dried using an inlettemperature of 225° C. and a dryer exit temperature of 76° C. Theprotein concentration in the resulting powder was 20.3 wt %, and the drysolids content was 93.2 wt %.

Example 7 Effect of Added Solvent on Peracetic Acid Production byThermotoga neapolitana Perhydrolase

A first mixture of 90.0 g of deionized water, 0.350 g of TURPINAL® SL((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt % in water;Thermphos International, Hague, Netherlands), and 3.20 g of 30 wt %hydrogen peroxide in water was adjusted to pH 7.2 with 50% aqueoussodium hydroxide, and the final weight of the mixture adjusted to 100.0g with deionized water. A second mixture of 55.76 g triacetin, 4.20 g ofsodium bicarbonate, 2.50 g of CAB-O-SIL® M5 fumed silica (Cabot, Boston,Mass.), 0.270 g of spray-dried Thermotoga neapolitana perhydrolase(Example 6), and 37.43 g of one organic solvent selected from the groupconsisting of tripropylene glycol methyl ether (DOWANOL® PM; DowChemical Corporation, Midland, Mich.), dipropylene glycol methyl ether(DOWANOL® DPM), propylene glycol methyl ether (DOWANOL® PM), diethyleneglycol butyl ether (DOWANOL® DB), dipropylene glycol (DOWANOL® DPG),triethylene glycol, 1,2-propanediol, N-ethyl-2-pyrroldinone,isopropanol, ethanol, ethyl lactate, and 1,3-propanediol was prepared. A1.0 g aliquot of the second mixture was removed with rapid stirring (tosuspend the undissolved solids) and mixed with 9.00 mL of the firstmixture of hydrogen peroxide, and TURPINAL® SL in water (pH 7.2) wasadded thereto with stirring at 25° C.; the resulting mixture contained255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL ofspray-dried perhydrolase. A control reaction for each solvent was alsorun to determine the concentration of peracetic acid produced bychemical perhydrolysis of triacetin by hydrogen peroxide in the absenceof added protein.

Determination of the concentration of peracetic acid in the reactionmixtures was performed according to the method described by Karst etal., supra. Aliquots (0.040 mL) of the reaction mixture were removed atpredetermined times and mixed with 0.960 mL of 5 mM phosphoric acid inwater; adjustment of the pH of the diluted sample to less than pH 4immediately terminated the reaction. The resulting solution was filteredusing an ULTRAFREE® MC-filter unit (30,000 Normal Molecular Weight Limit(NMWL), Millipore Corp., Billerica, Mass.; cat #UFC3LKT 00) bycentrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of theresulting filtrate was transferred to 1.5-mL screw cap HPLC vial(Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing 0.300mL of deionized water, then 0.100 mL of 20 mM MTS(methyl-p-tolyl-sulfide) in acetonitrile was added, the vials capped,and the contents briefly mixed prior to a 10 min incubation at ca. 25°C. in the absence of light. To each vial was then added 0.400 mL ofacetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP, 40mM) in acetonitrile, the vials re-capped, and the resulting solutionmixed and incubated at ca. 25° C. for 30 min in the absence of light. Toeach vial was then added 0.100 mL of 10 mM N,N-diethyl-m-toluamide(DEET; HPLC external standard) and the resulting solution analyzed byHPLC as described below.

HPLC Method:

Supelco Discovery C8 column (10-cm×4.0-mm, 5 μm) (cat. #569422-U)w/precolumn Supelco Supelguard Discovery C8 (Sigma-Aldrich; cat#59590-U); 10 microliter injection volume; gradient method with CH₃CN(Sigma-Aldrich; #270717) and deionized water at 1.0 mL/min and ambienttemperature:

Time (min:sec) (% CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00(stop) 40

The peracetic acid concentrations produced in 0.5 min, 1 min, 2 min, 5min and 10 min for the reactions described above are listed in Table 5,below.

TABLE 5 Dependence of peracetic acid (PAA) concentration on solventaddition using triacetin (255 mM), hydrogen peroxide (254 mM) and 0.055mg/mL of spray-dried Thermotoga neapolitana perhydrolase. Enzyme PAA(ppm) Solvent (μg/mL) 0.5 min 1 min 2 min 5 min 10 min DOWANOL ® PM 0 3954 52 81 137 DOWANOL ® DPM 0 136 41 106 386 ND DOWANOL ® TPM 0 23 25 11193 180 DOWANOL ® DB 0 107 102 105 157 218 DOWANOL ® DPG 0 19 40 101 156207 Triethylene glycol 0 36 53 110 76 307 1,2-propanediol 0 101 96 122226 347 N-ethyl-2- 0 37 49 60 77 133 pyrroldinone isopropanol 0 70 13147 150 242 ethanol 0 68 33 150 356 479 ethyl lactate 0 88 91 98 121 1371,3-propanediol 0 54 48 48 62 107 DOWANOL ® PM 55 355 1327 1632 31565378 DOWANOL ® DPM 55 846 972 1587 3209 4494 DOWANOL ® TPM 55 439 5391303 2710 3740 DOWANOL ® DB 55 475 827 1719 3222 4863 DOWANOL ® DPG 55583 769 1211 2784 4522 Triethylene glycol 55 325 834 1634 3229 51161,2-propanediol 55 507 903 1428 2921 4364 N-ethyl-2- 55 243 837 14703033 4839 pyrroldinone isopropanol 55 326 656 1175 2229 2860 ethanol 55408 584 1109 2235 2858 ethyl lactate 55 180 337 5736 1420 25541,3-propanediol 55 163 269 510 1086 1657

To demonstrate the stability of the spray-dried enzyme in a mixture oftriacetin and an organic solvent, the mixtures of triacetin, sodiumbicarbonate, CAB-O-SIL® M5 (Cabot), spray-dried Thermotoga neapolitanaperhydrolase (Example 6), and either tripropylene glycol methyl ether(DOWANOL® TPM) or 1,2-propanediol described above were stored for 24 hat ambient temperature, then a 1.0 g aliquot of each of these mixtureswas removed with rapid stirring (to suspend the undissolved solids) andmixed with 9.0 mL of a freshly prepared (as described above) mixture ofhydrogen peroxide and TURPINAL® SL in water (pH 7.2) with stirring at25° C.; the resulting mixture contained 255 mM triacetin, 254 mMhydrogen peroxide and 0.055 mg protein/mL of spray-dried perhydrolase. Acontrol reaction for each solvent was also run to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added protein.Determination of the concentration of peracetic acid in the reactionmixtures was performed according to the method described by Karst et al.(Table 6).

TABLE 6 Stability of perhydrolase in triacetin/solvent suspension,measured in reactions containing triacetin (255 mM) and hydrogenperoxide (254 mM). Enzyme PAA (ppm) Solvent (μg/mL) 0.5 min 1 min 2 min5 min 10 min DOWANOL ® TPM 0 0 95 58 172 276 1,2-propanediol 0 16 38 35171 397 DOWANOL ® TPM 55 386 557 1078 2014 2717 1,2-propanediol 55 566768 1467 3093 4649

Example 8 Comparison of Peracetic Acid Production by ThermotogaNeapolitana Perhydrolase in Presence or Absence of Added Solvent

A first mixture of 40.0 g of deionized water, 0.1575 g of TURPINAL® SL((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt % in water;Thermphos International), and 1.44 g of 30 wt % hydrogen peroxide inwater was adjusted to pH 7.2 with 50% aqueous sodium hydroxide, and thefinal weight of the mixture adjusted to 46.87 g with deionized water. Asecond mixture of 2.78 g triacetin, 0.210 g of sodium bicarbonate, 0.125g of CAB-O-SIL® M5 (Cabot) and 0.0135 g of spray-dried Thermotoganeapolitana perhydrolase (Example 6) was prepared, and the first mixtureof hydrogen peroxide and TURPINAL® SL in water (pH 7.2) was added to thesecond mixture with stirring at 25° C.; the resulting mixture contained255 mM triacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL ofspray-dried perhydrolase. Determination of the concentration ofperacetic add in the reaction mixtures was performed according to themethod described by Karst et al., supra. A control reaction was also runto determine the concentration of peracetic acid produced by chemicalperhydrolysis of triacetin by hydrogen peroxide in the absence of addedperhydrolase.

The reaction described above was repeated, where 1.872 g of eitherpropylene glycol monomethyl ether (DOWANOL® PM) or dipropyleneglycolmonomethyl ether (DOWANOL® DPM), was substituted for an equivalentweight of water in the reaction mixture. A first mixture of 40.0 g ofdeionized water, 0.175 g of TURPINAL® SL, and 1.60 g of 30 wt % hydrogenperoxide in water was adjusted to pH 7.2 with 50% aqueous sodiumhydroxide, and the final weight of the mixture adjusted to 50.0 g withdeionized water. A second mixture of 2.78 g triacetin, 1.872 g of eitherpropylene glycol monomethyl ether (DOWANOL® PM) or dipropyleneglycolmonomethyl ether (DOWANOL® DPM), 0.210 g of sodium bicarbonate, 0.125 gof CAB-O-SIL® M5 (Cabot) and 0.0135 g of spray-dried Thermotoganeapolitana perhydrolase (Example 6) was prepared, and 45.0 g of thefirst mixture of hydrogen peroxide and TURPINAL® SL in water (pH 7.2)was added to the second mixture with stirring at 25° C.; the resultingmixture (pH 6.5) contained 255 mM triacetin, 254 mM hydrogen peroxideand 0.055 mg protein/mL of spray-dried perhydrolase. A control reactionwas also run to determine the concentration of peracetic acid producedby chemical perhydrolysis of triacetin by hydrogen peroxide in theabsence of added extract protein. The peracetic acid concentrationsproduced in 0.5 min, 1 min, 2 min, 5 min and 10 min for the threereactions described above are listed in Table 7, below.

TABLE 7 Dependence of peracetic acid (PAA) concentration on solventaddition using triacetin (255 mM), hydrogen peroxide (254 mM) and 55μg/mL of spray-dried Thermotoga neapolitana perhydrolase. Enzyme PAA(ppm) Solvent (μg/mL) 0.5 min 1 min 2 min 5 min 10 min none 0 ND ND NDND ND DOWANOL ® PM 0 89 90 205 318 498 DOWANOL ® DPM 0 104 178 184 373535 none 55 629 1359 2020 4274 6019 DOWANOL ® PM 55 807 1390 2331 44395917 DOWANOL ® DPM 55 787 1373 2566 5122 6528

Example 9 Use of Solvent for In Situ Peracid Generation Using aTwo-Compartment Spray-Bottle Compared to Stirred Reactions

A first mixture of 100 g of 0.20 M sodium citrate buffer containing 2000ppm TURPINAL® SL ((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt %in water; Thermphos International), 280 g of deionized water, and 5.20 gof 30 wt % hydrogen peroxide in water was adjusted to pH 7.2 with 50%aqueous sodium hydroxide, and the final weight of the mixture adjustedto 400 g with deionized water. A second mixture was separately prepared,containing 83.4 g of triacetin, 3.75 g of CAB-O-SIL® M5 (Cabot), 0.750 gof spray-dried Thermotoga neapolitana perhydrolase (Example 6), and 62.1g of a single

solvent selected from: propylene glycol methyl ether (DOWANOL° PM),tripropylene glycol methyl ether (DOWANOL® TPM), diethylene glycolmethyl ether (DOWANOL® DM), propylene glycol n-butyl ether (DOWANOL®PNB), propylene glycol n-propyl ether (DOWANOL® PnP), propylene glycolmonomethyl ether acetate (DOWANOL® PMA), dipropylene glycol, ethanol,isopropanol, and 1,2-propanediol. In a first reaction at 25° C., 1.0 gof the first mixture was stirred with 9.0 g of the second mixture forthe first 30-60 seconds of the reaction (reaction pH of 6.5-6.0), andsamples were withdrawn and analyzed for peracetic acid production; theresulting reaction mixture contained 255 mM triacetin, 103 mM hydrogenperoxide and 100 μg protein/mL of spray-dried perhydrolase.Determination of the concentration of peracetic acid in the reactionmixtures (TABLE 8, below) was performed according to the methoddescribed by Karst et al., supra. A control reaction was also run todetermine the concentration of peracetic acid produced by chemicalperhydrolysis of triacetin by hydrogen peroxide in the absence of addedperhydrolase.

The first mixture and second mixture prepared as described above wereeach separately charged to one of the two compartments of atwo-compartment spray bottle (Custom Dual-Liquid Variable-Ratio Sprayer,Model DLS 200, manufactured by Take5 (Rogue River, Oreg.)), where thebottle was set up to spray a mixture of 9 parts by weight of the firstmixture with 1 part by weight of the second mixture. The two mixtureswere sprayed into a 12.5 cm diameter crystallizing dish, and theresulting reaction mixture (reaction pH of 6.5-6.0) contained 255 mMtriacetin, 100 mM hydrogen peroxide and 0.100 mg protein/mL ofspray-dried perhydrolase. The sprayed reaction mixture was sampled atpredetermined times and analyzed for peracetic acid (TABLE B, below)according to the method described by Karst et al., supra.

TABLE 8 Dependence of peracetic acid (PAA) concentration on solventaddition using triacetin (255 mM), hydrogen peroxide (103 mM) and 0.100mg/mL of spray- dried Thermotoga neapolitana perhydrolase in stirredbatch reactions and in a sprayed two-component mixture. Enzyme PAA (ppm)Solvent (μg/mL) 20 sec 40 sec 60 sec 120 sec 300 sec 600 sec DOWANOL ®PM, 0 101 106 106 82 90 166 stirred reaction DOWANOL ® PM, 100 319 587622 648 889 976 stirred reaction DOWANOL ® PM, 100 375 454 515 671 873994 sprayed reaction DOWANOL ® TPM, 0 0 72 19 25 44 69 stirred reactionDOWANOL ® TPM, 100 445 548 726 980 1378 1560 stirred reaction DOWANOL ®TPM, 100 433 575 1385 806 1089 1250 sprayed reaction DOWANOL ® DM, 0 287261 287 261 255 234 stirred reaction DOWANOL ® DM, 100 667 875 927 14101640 1876 stirred reaction DOWANOL ® DM, 100 540 613 866 914 1112 1276sprayed reaction DOWANOL ® PNB, 0 76 70 40 58 0 11 stirred reactionDOWANOL ® PNB, 100 344 488 654 932 1166 1357 stirred reaction DOWANOL ®PNB, 100 394 514 586 715 963 1141 sprayed reaction DOWANOL ® PnP, 0 173163 223 215 213 253 stirred reaction DOWANOL ® PnP, 100 611 716 857 12771468 1516 stirred reaction DOWANOL ® PnP, 100 371 657 737 928 1090 1195sprayed reaction DOWANOL ® PMA, 0 0 0 14 0 128 166 stirred reactionDOWANOL ® PMA, 100 335 510 756 1218 2178 3132 stirred reaction DOWANOL ®PMA, 100 541 745 1042 1472 ND 3236 sprayed reaction dipropylene glycol,0 26 54 73 79 40 38 stirred reaction dipropylene glycol, 100 318 539 7081423 1241 946 stirred reaction dipropylene glycol, 100 371 414 464 618756 863 sprayed reaction ethanol, 0 144 184 152 161 167 170 stirredreaction ethanol, 100 398 553 694 919 1227 1311 stirred reactionethanol, 100 504 677 685 766 968 1125 sprayed reaction isopropanol, 0149 167 180 207 180 236 stirred reaction isopropanol, 100 564 691 7831114 1395 1533 stirred reaction isopropanol, 100 621 767 882 1014 12391435 sprayed reaction 1,2-propanediol, 0 32 14 19 33 ND 108 stirredreaction 1,2-propanediol, 100 427 665 921 1485 1941 3466 stirredreaction 1,2-propanediol, 100 376 554 704 1376 1873 2517 sprayedreaction cyclohexanone, 0 136 133 153 138 152 114 stirred reactioncyclohexanone, 100 97 153 185 351 329 459 stirred reactioncyclohexanone, 100 128 196 338 368 416 489 sprayed reaction

Example 10 Peroxycarboxylic Acid Production Using Thermotoga maritimaPerhydrolase as an Enzyme Catalyst

Cloning and expression of perhydrolase from Thermotoga maritima isaccomplished in accordance with the methods described in precedingExamples 1-4. Fermentation of bacterial transformants expressingThermotoga maritima perhydrolase is performed in accordance withpreceding Example 5, and preparation of spray-dried Thermotoga maritimaperhydrolase is accomplished using methods described in Example 6.Additional information regarding techniques for cloning, expressing, andpreparation of Thermotoga maritima perhydrolase is available inPublished U.S. Patent Application No. 2009/0005590; herein incorporatedby reference.

A comparison of peracetic acid production by Thermotoga maritimaperhydrolase in the presence and absence of added solvent is performed.A first mixture of 40.0 g of deionized water, 0.1575 g of TURPINAL® SL((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt % in water;Thermphos International), and 1.44 g of 30 wt % hydrogen peroxide inwater is adjusted to pH 7.2 with 50% aqueous sodium hydroxide, and thefinal weight of the mixture adjusted to 46.87 g with deionized water. Asecond mixture of 2.78 g triacetin, 0.210 g of sodium bicarbonate, 0.125g of CAB-O-SIL® M5 (Cabot) and 0.0135 g of spray-dried Thermotogamaritima perhydrolase is prepared, and the first mixture of hydrogenperoxide and TURPINAL® SL in water (pH 7.2) is added to the secondmixture with stirring at 25° C.; the resulting mixture containing 255 mMtriacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL ofspray-dried perhydrolase. Determination of the concentration ofperacetic acid in the reaction mixtures is performed according to themethod described by Karst et al., supra. A control reaction is also runto determine the concentration of peracetic acid produced by chemicalperhydrolysis of triacetin by hydrogen peroxide in the absence of addedperhydrolase.

The reaction described above is repeated, where 1.872 g of eitherpropylene glycol monomethyl ether (DOWANOL® PM) or dipropyleneglycolmonomethyl ether (DOWANOL® DPM), is substituted for an equivalent weightof water in the reaction mixture. A first mixture of 40.0 g of deionizedwater, 0.175 g of TURPINAL® SL, and 1.60 g of 30 wt % hydrogen peroxidein water is adjusted to pH 7.2 with 50% aqueous sodium hydroxide, andthe final weight of the mixture adjusted to 50.0 g with deionized water.A second mixture of 2.78 g triacetin, 1.872 g of either propylene glycolmonomethyl ether (DOWANOL® PM) or dipropyleneglycol monomethyl ether(DOWANOL® DPM), 0.210 g of sodium bicarbonate, 0.125 g of CAB-O-SIL® M5(Cabot) and 0.0135 g of spray-dried Thermotoga maritima perhydrolase isprepared, and 45.0 g of the first mixture of hydrogen peroxide andTURPINAL® SL in water (pH 7.2) is added to the second mixture withstirring at 25° C.; the resulting mixture (pH 6.5) contained 255 mMtriacetin, 254 mM hydrogen peroxide and 0.055 mg protein/mL ofspray-dried perhydrolase. A control reaction is also run to determinethe concentration of peracetic acid produced by chemical perhydrolysisof triacetin by hydrogen peroxide in the absence of added extractprotein. The peracetic acid concentrations produced in 0.5 min, 1 min, 2min, 5 min and 10 min for the three reactions described above aremeasured and recorded.

Example 11 Use of Solvent for In Situ Peracid Generation UsingTwo-Compartment Spray Device Compared to Stirred Reaction and UsingThermotoga maritima Perhydrolase

A first mixture of 100 g of 0.20 M sodium citrate buffer containing 2000ppm TURPINAL® SL ((1-hydroxy-1-phosphonoethyl)phosphonic acid, 60 wt %in water; Thermphos International), 280 g of deionized water, and 5.20 gof 30 wt % hydrogen peroxide in water is adjusted to pH 7.2 with 50%aqueous sodium hydroxide, and the final weight of the mixture adjustedto 400 g with deionized water. A second mixture is separately prepared,containing 83.4 g of triacetin, 3.75 g of CAB-O-SIL® M5 (Cabot), 0.750 gof spray-dried Thermotoga maritima perhydrolase (Example 10), and 62.1 gof a single solvent selected from propylene glycol methyl ether(DOWANOL® PM), tripropylene glycol methyl ether (DOWANOL® TPM),diethylene glycol methyl ether (DOWANOL®DM), propylene glycol n-butylether (DOWANOL® PNB), propylene glycol n-propyl ether (DOWANOL® PnP),propylene glycol monomethyl ether acetate (DOWANOL® PMA), dipropyleneglycol, ethanol, isopropanol, and 1,2-propanediol. In a first reactionat 25° C., 1.0 g of the first mixture is stirred with 9.0 g of thesecond mixture for the first 30-60 seconds of the reaction (reaction pHof 6.5-6.0), and samples are withdrawn and analyzed for peracetic acidproduction; the resulting reaction mixture containing 255 mM triacetin,103 mM hydrogen peroxide and 100 μg protein/mL of spray-driedperhydrolase. Determination of the concentration of peracetic acid inthe reaction mixtures is performed according to the method described byKarst et al., supra. A control reaction is also run to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added perhydrolase.

The first mixture and second mixture prepared as described above areeach separately charged to one of the two compartments of atwo-compartment spray bottle (Custom Dual-Liquid Variable-Ratio Sprayer,Model DLS 200, manufactured by Take5 (Rogue River, Oreg.)), where thebottle is set up to spray a mixture of 9 parts by weight of the firstmixture with 1 part by weight of the second mixture. The two mixturesare sprayed into a 12.5 cm diameter crystallizing dish, and theresulting reaction mixture (reaction pH of 6.5-6.0) containing 255 mMtriacetin, 100 mM hydrogen peroxide and 0.100 mg protein/mL ofspray-dried perhydrolase. The sprayed reaction mixture is sampled atpredetermined times and analyzed for peracetic acid according to themethod described by Karst et al., supra.

Example 12 Exemplary Two-Component System

One example of a two-component in situ peracid disinfectant formulationis described below.

mol/L grams Component A triacetin 0.100 21.82 T. neapolitanaperhydrolase/excipient 0.20 sodium bicarbonate 0.050 4.20 Component BH₂O₂ (30 wt %): 0.100 11.33 TURPINAL ® SL (60 wt %, 0.1% final) 1.67water (deionized) 960.78 Total weight (grams) 1000.00

For the two-component in situ peracid disinfectant formulation describedabove, Component A comprises ca. 2.6 wt % of the combined weight ofComponents A and B, and the weight ratio of Component B to Component Ais ca. 38:1. In certain applications for a two-component in situ peraciddisinfectant formulation, it may be desirable for the ratio of ComponentB to Component A to be within a range of from 1:1 to 10:1, where from 10parts to 1 part (by weight) of Component B is mixed with one part (byweight) of Component A to produce a peracid at a concentrationefficacious for disinfection. For example, in a first application atwo-compartment spray bottle such as a dual-liquid fixed ratio sprayer(Model DLS100, Take5) or a dual-liquid variable ratio sprayer (ModelDLS200, Take5) is utilized, where a maximum ratio of Component B toComponent A of 10:1 is employed. In a second application, a singlebottle containing two separate compartments separated by a breakableseal is employed, where the ratio of the volume of the two separatecompartments is 1:1, or 5:1 or 10:1. In each of these applications, thetwo-component formulation cannot be mixed at the desired ratio ofComponent A to Component B to provide the desired concentration ofreactants and final concentration of products.

Example 13 Perhydrolysis of Propylene Glycol Diacetate or EthyleneGlycol Diacetate Using Bacillus subtilis ATCC® 31954™ Perhydrolase

A homogenate of a transformant expressing wild-type perhydrolase fromBacillus subtilis ATCC® 31954™ (KLP18/pSW194) was prepared from asuspension of cell paste (20 wt % wet cell weight) in 0.05 M potassiumphosphate buffer (pH 7.0) containing dithiothreitol (1 mM). The crudehomogenate was centrifuged to remove cellular debris, producing aclarified cell extract that was heat-treated at 65° C. for 30 min. Theresulting mixture was centrifuged, and the heat-treated supernatantconcentrated on a 30K MWCO (molecular weight cutoff) membrane to aconcentration of 32 mg/mL total dissolved solids; a SOS-PAGE of theclarified, heat-treated cell extract indicated that the perhydrolase wasat least 85-90% pure. To this concentrate was then added 2.06 grams ofNaH₂PO₄ and 1.17 grams Na₂HPO₄ per gram of solids was added to thisconcentrate to produce an approximate 3:1 ratio (wt/wt) of phosphatebuffer to heat-treated cell extract protein. This solution was dilutedby 30 wt % with deionized water, then spray-dried (180° C. inlettemperature, 70° C. exit temperature) using a Buchi B-290 laboratoryspray dryer); the resulting spray-dried powder contained 25.5 wt %protein (Bradford protein assay) and was 94.3 wt % dry solids.

Reactions (10 mL total volume) were run at 23° C. in 50 mM sodiumbicarbonate buffer (initial pH 7.2) containing propylene glycoldiacetate (PGDA) or ethylene glycol diacetate (EGDA), hydrogen peroxide(100 mM) and 123 μg/mL of a heat-treated extract protein from thespray-dried E. coli KLP18/pSW194 (expressing Bacillus subtilis ATCC®31954™ wild-type perhydrolase) (prepared as described above). A controlreaction for each reaction condition was run to determine theconcentration of peracetic acid produced by chemical perhydroiysis oftriacetin by hydrogen peroxide in the absence of added heat-treatedextract protein. The reactions were sampled at 1, 5, and 30 minutes andthe samples analyzed for peracetic acid using the Karst derivatizationprotocol (Karst at al., supra); aliquots (0.040 mL) of the reactionmixture were removed and mixed with 0.960 mL of 5 mM phosphoric acid inwater; adjustment of the pH of the diluted sample to less than pH 4immediately terminated the reaction. The resulting solution was filteredusing an ULTRAFREE® MC-filter unit (30,000 Normal Molecular Weight Limit(NMWL), Millipore cat #UFC3LKT 00) by centrifugation for 2 min at 12,000rpm. An aliquot (0.100 mL) of the resulting filtrate was transferred to1,5-mL screw cap HPLC vial (Agilent Technologies, Palo Alto, Calif.;#5182-0715) containing 0.300 mL of deionized water, then 0.100 mL of 20mM MTS (methyl-p-tolyl-sulfide) in acetonitrile was added, the vialscapped, and the contents briefly mixed prior to a 10 min incubation atca 25° C. in the absence of light. To each vial was then added 0.400 mLof acetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP,40 mM) in acetonitrile, the vials re-capped, and the resulting solutionmixed and incubated at ca. 25° C. for 30 min in the absence of light. Toeach vial was then added 0.100 mL of 10 mM N,N-diethyl-m-toluamide(DEET; HPLC external standard) and the resulting solution analyzed byHPLC. The peracetic acid concentrations produced in 1 min, 5 min and 30min are listed in Table 9.

TABLE 9 Peracetic acid (PAA) concentration produced in reactionsutilizing propylene glycol diacetate (PGDA) or ethylene glycol diacetate(EGDA) and hydrogen peroxide (100 mM) in sodium bicarbonate buffer (50mM, initial pH 7.2) at 23° C. using 123 μg/mL of heat-treated extractprotein from E. coli KLP18/pSW194 (Bacillus subtilis ATCC ® 31954 ™perhydrolase). PAA, PAA, PAA, perhydrolase substrate 1 min 5 min 30 min(50 μg/mL) (100 mM) (ppm) (ppm) (ppm) no enzyme (control) PGDA 0 64 241B. subtilis ATCC ® 31954 PGDA 666 781 815 no enzyme (control) EGDA 0 18141 B. subtilis ATCC ® 31954 EGDA 747 931 963

Example 14 Perhydrolysis of Propylene Glycol Diacetate or EthyleneGlycol Diacetate Using T. maritima and T. neapolitana Wild-Type andVariant Perhydrolases

Cell extracts of transformants expressing Thermotoga neapolitanawild-type perhydrolase (KLP18/pSW196), Thermotoga neapolitana C277Svariant perhydrolase (KLP18/pSW196/C277S), Thermotoga neapolitana C277Tvariant perhydrolase (KLP18/pSW196/C277T), Thermotoga maritima wild-typeperhydrolase (KLP18/pSW228), Thermotoga maritima C277S variantperhydrolase (KLP18/pSW228/C277S), and Thermotoga maritima C277T variantperhydrolase (KLP18/pSW228/C277T) were each prepared by passing asuspension of cell paste (20 wt % wet cell weight) in 0.05 M potassiumphosphate buffer (pH 7.0) containing dithiothreitol (1 mM) twice througha French press having a working pressure of 16,000 psi (˜110 MPa). Thelysed cells were centrifuged for 30 minutes at 12,000×g, producing aclarified cell extract that was assayed for total soluble protein(Bradford assay). The supernatant was heated at 75° C. for 20 minutes,followed by quenching in an ice bath for 2 minutes. Precipitated proteinwas removed by centrifugation for 10 minutes at 11,000×g, SDS-PAGE ofthe resulting heat-treated extract protein supernatant indicated thatthe CE-7 enzyme comprised approximately 85-90% of the total protein inthe preparation. The heat-treated extract protein supernatant was frozenin dry ice and stored at −80° C. until use

A first set of reactions (10 mL total volume) were run at 20° C. in 10mM sodium bicarbonate buffer (initial pH 8.1) containing propyleneglycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (100 mM),hydrogen peroxide (100 mM) and 25 μg/mL of heat-treated extract proteinfrom one of E. coli KLP18/pSW195 (Thermotoga neapolitana wild-typeperhydrolase), E. coli KLP18/pSW196/C277S (Thermotoga neapolitana C277Svariant perhydrolase), E. coli KLP18/pSW196/C277T (Thermotoganeapolitana C277T variant perhydrolase), E. coli KLP18/pSW228(Thermotoga maritima wild-type perhydrolase), E. coli KLP18/pSW228/C277S(Thermotoga maritima C277S variant perhydrolase), and E. coliKLP18/pSW228/C277T (Thermotoga maritima C277T variant perhydrolase)(prepared as described above). A control reaction for each reactioncondition was run to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The reactions were sampled at 1,5, and 30 minutes and the samples analyzed for peracetic acid using theKarst derivatization protocol (Karst et al., supra) and HPLC analyticalmethod (supra). The peracetic acid concentrations produced in 1 min, 5min and 30 min are listed in Table 10.

TABLE 10 Peracetic acid (PAA) concentration produced utilizing T.maritima and T. neapolitana wild-type and variant perhydrolases inreactions at 20° C. in sodium bicarbonate buffer (10 mM, initial pH 8.1)containing propylene glycol diacetate (PGDA) (100 mM) or ethylene glycoldiacetate (EGDA) (100 mM), hydrogen peroxide (100 mM) and 25 μg/mL ofheat-treated extract protein. substrate PAA, PAA, PAA, sub- conc. H₂O₂ 1min 5 min 30 min perhydrolase strate (mM) (mM) (ppm) (ppm) (ppm) noenzyme PGDA 100 100 0 15 165 (control) T. maritima PGDA 100 100 534 11041695 WT T. maritima PGDA 100 100 647 1320 1864 C277S T. maritima PGDA100 100 656 1174 1418 C277T T. neapolitana PGDA 100 100 513 1052 1946 WTT. neapolitana PGDA 100 100 875 1327 1707 C277S T. neapolitana PGDA 100100 724 1325 1864 C277T no enzyme EGDA 100 100 0 70 229 (control) T.maritima EGDA 100 100 765 1182 1595 WT T. maritima EGDA 100 100 725 12401724 C277S T. maritima EGDA 100 100 802 1218 1734 C277T T. neapolitanaEGDA 100 100 603 1132 1643 WT T. neapolitana EGDA 100 100 680 1305 1698C277S T. neapolitana EGDA 100 100 688 1164 1261 C277T

A second set of reactions (10 mL total volume) were run at 20° C. in 10mM sodium bicarbonate buffer (initial pH 8.1) containing propyleneglycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (2 mM),hydrogen peroxide (10 mM) and 10 μg/mL of heat-treated extract proteinfrom one of E. coli KLP18/pSW196 (Thermotoga neapolitana wild-typeperhydrolase), E. coli KLP18/pSW196/C277S (Thermotoga neapolitana C277Svariant perhydrolase), E. coli KLP18/pSW196/C277T (Thermotoganeapolitana C277T variant perhydrolase), E. coli KLP18/pSW228(Thermotoga maritima wild-type perhydrolase), E. coli KLP18/pSW228/C277S(Thermotoga maritima C277S variant perhydrolase), and E. coliKLP18/pSW228/C277T (Thermotoga maritima C277T variant perhydrolase)(prepared as described above). A control reaction for each reactioncondition was run to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The reactions were sampled at 5minutes and the samples analyzed for peracetic acid using the Karstderivatization protocol (Karst et al., supra) and HPLC analytical method(supra). The peracetic acid concentrations produced in 5 min are listedin Table 11.

TABLE 11 Peracetic acid (PAA) concentration produced utilizing T.maritima and T. neapolitana wild-type and variant perhydrolases inreactions at 20° C. in sodium bicarbonate buffer (10 mM, initial pH 8.1)containing propylene glycol diacetate (PGDA) (2 mM) or ethylene glycoldiacetate (EGDA) (2 mM), hydrogen peroxide (10 mM) and 10 μg/mL ofheat-treated extract protein. substrate PAA, conc. H₂O₂ 5 minperhydrolase substrate (mM) (mM) (ppm) no enzyme (control) PGDA 2 10 3.6T. maritima WT PGDA 2 10 5.0 T. maritima C277S PGDA 2 10 7.2 T. maritimaC277T PGDA 2 10 7.9 T. neapolitana WT PGDA 2 10 5.7 T. neapolitana C277SPGDA 2 10 7.9 T. neapolitana C277T PGDA 2 10 3.9 no enzyme (control)EGDA 2 10 3.3 T. maritima WT EGDA 2 10 9.9 T. maritima C277S EGDA 2 1013.6 T. maritima C277T EGDA 2 10 22.9 T. neapolitana WT EGDA 2 10 6.6 T.neapolitana C277S EGDA 2 10 18.4 T. neapolitana C277T EGDA 2 10 20.2

Example 15 Expression of Thermotoga neapolitana Acetyl Xylan EsteraseVariants in E. coil KLP18

Plasmids comprising acetyl xylan esterase mutations as described inco-owned, co-filed, and copending U.S. patent application AttorneyDocket No. CL4392 US NA were prepared from wild type Thermotoganeapolitana perhydrolase (SEQ ID NO: 14) by substituting at amino acidresidue position 277 an Ala, Val, Ser, or Thr (SEQ ID NO: 73). Theplasmids were used to transform E. coli KLP18 (Example 3). Transformantswere plated onto LB-ampicillin (100 mg/mL) plates and incubatedovernight at 37° C. Cells were harvested from a plate using 2.5 mL LBmedia supplemented with 20% (v/v) glycerol, and 1.0 mL aliquots of theresulting cell suspension frozen at −80° C. One mL of the thawed cellsuspension was transferred to a 1-L APPLIKON® Bioreactor (Applikon®Biotechnology, Foster City, Calif.) with 0.7 L medium containing KH₂PO₄(5.0 g/L), FeSO₄ heptahydrate (0.05 g/L), MgSO₄ heptahydrate (1.0 g/L),sodium citrate dihydrate (1.90 g/L), yeast extract (Amberex 695, 5.0g/L), Biospumex 153K antifoam (0.25 mL/L, Cognis Corporation), NaCl (1.0WO, CaCl₂ dihydrate (0.1 g/L), and NIT trace elements solution (10mL/L). The trace elements solution contained citric acid monohydrate (10g/L), MnSO₄ hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L),ZnSO₄ heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization additions included glucosesolution (50% w/w, 6.5 g) and ampicillin (25 mg/mL) stock solution (2.8mL). Glucose solution (50% w/w) was also used for fed batch, Glucosefeed was initiated 40 min after glucose concentration decreased below0.5 g/L, starting at 0.03 g feed/min and increasing progressively eachhour to 0.04, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.12, and 0.14 g/minrespectively; the rate remaining constant afterwards. Glucoseconcentration in the medium was monitored, and if the concentrationexceeded 0.1 g/L the feed rate was decreased or stopped temporarily.Induction was initiated at OD₅₅₀=50 with addition of 0.8 mL IPTG (0.05M). The dissolved oxygen (DO) concentration was controlled at 25% of airsaturation, first by agitation (400-1000 rpm), and following by aeration(0.5-2 slpm). The temperature was controlled at 37° C., and the pH wascontrolled at 6.8; NH₄OH (29% w/w) and H₂SO₄ (20% w/v) were used for pHcontrol. The cells were harvested by centrifugation (5,000×g for 15minutes) at 20 h post IPTG addition. A cell culture of E. coliKLP18/pSW196 (Thermotoga neapolitana wild-type perhydrolase) was grownas described in Example 2.

Example 16 Preparation of Cell Lysates Containing Semi-Purified WildType T. Neapolitana Acetyl Xylan Esterase or T. Neapolitana VariantAcetyl Xylan Esterases

A cell culture of E. coli KLP18/pSW196 (Thermotoga neapolitana wild-typeperhydrolase) was grown as described in Example 5. The resulting cellpaste was resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0supplemented with 1.0 mM DTT. Resuspended cells were passed through aFrench pressure cell twice to ensure>95% cell lysis, Lysed cells werecentrifuged for 30 minutes at 12,000×g, and the supernatant was heatedat 75° C. for 20 minutes, followed by quenching in an ice bath for 2minutes. Precipitated protein was removed by centrifugation for 10minutes at 11,000×g. SOS-PAGE indicated that the CE-7 enzyme comprisedapproximately 85-90% of the total protein in the heat-treated extractsupernatant.

Cell cultures of E. coli KLP18/pSW196/C2778 (Thermotoga neapolitanaC277S variant perhydrolase), E. coli KLP18/pSW196/C2771/(Thermotoganeapolitana C277V variant perhydrolase), E. coli KLP18/pSW196/C277A(Thermotoga neapolitana C277A variant perhydrolase), and E. coliKLP18/pSW196/C277T (Thermotoga neapolitana C277T variant perhydrolase)were each grown as described in Example 15. The resulting cell pasteswere resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0 supplementedwith 1.0 mM DTT. Resuspended cells were passed through a French pressurecell twice to ensure>95% cell lysis. Lysed cells were centrifuged for 30minutes at 12,000×g, and the supernatant was heated at 75° C. for 20minutes, followed by quenching in an ice bath for 2 minutes.Precipitated protein was removed by centrifugation for 10 minutes at11,000×g. SDS-PAGE indicated that the CE-7 enzyme comprisedapproximately 85-90% of the total protein in the heat-treated extractsupernatant.

Example 17 Specific Activity and Perhydrolysis/Hydrolysis Ratio of T.Neapolitana Acetyl Xylan Wild-Type Esterase and 0277 Esterase Variants

Reactions (40 mL total volume) were run at 25° C. in phosphate buffer(50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100mM) and one of the following acetyl xylan esterase variants: T.neapolitana C277S variant perhydrolase (0.010 mg/mL of heat-treatedextract total protein from E. coli KLP18/pSW196/C277S), T. neapolitanaC277T variant perhydrolase (0.010 mg/mL of heat-treated extract totalprotein from E. coli KLP18/pSW196/C277T), T. neapolitana C277A variantperhydrolase (0.0125 mg/mL of heat-treated extract total protein from E.coli KLP18/pSW196/C277A), and T. neapolitana C277V variant perhydrolase(0.0125 mg/mL of heat-treated extract total protein from E. coliKLP18/pSW196/C277V) (prepared as described in Example 16). Reactionswere stirred for only the first 30 seconds of reaction to initially mixthe reactants and enzyme.

A reaction was also run under identical conditions to that describedimmediately above using 0.050 mg/ml of heat-treated extract totalprotein isolated from E. coli KLP18/pSW196 (expressing Thermotoganeapolitana wild-type acetyl xylan esterase (Example 1)), where theheat-treated extract supernatant was prepared according to the procedureof Example 16.

Two samples from each of the reaction mixtures described above weresimultaneously withdrawn after the first minute of each reaction, andevery two minutes thereafter for fifteen minutes, where one of the twosamples was analyzed for peracetic acid, and the second sample wasanalyzed for total acetic acid produced from both enzymatic hydrolysisof triacetin and from subsequent conversion of peracetic acid in sampleto acetic acid by reaction with methyl-p-tolyl sulfide (MTS, see below).

Measurement of the rate of peracetic acid production in the reactionmixture was performed using a modification of the method described byKarst et al., supra. A sample (0.040 mL) of the reaction mixture wasremoved at a predetermined time and immediately mixed with 0.960 mL of 5mM phosphoric acid in water to terminate the reaction by adjusting thepH of the diluted sample to less than pH 4. The resulting solution wasfiltered using an ULTRAFREE® MC-filter unit (30,000 Normal MolecularWeight Limit (NMWL), Millipore Corp., Billerica, Mass.; cat #UFC3LKT 00)by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 ml) of theresulting filtrate was transferred to a 1.5-mL screw cap HPLC vial(Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing 0.300mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-tolylsulfide) in acetonitrile was added, the vial capped, and the contentsbriefly mixed prior to a 10 min incubation at ca. 25° C. in the absenceof light. To the vial was then added 0.400 mL of acetonitrile and 0.100mL of a solution of triphenylphosphine (TPP, 40 mM) in acetonitrile, thevial recapped, and the resulting solution mixed and incubated at ca. 25°C. for 30 min in the absence of light. To the vial was then added 0.100mL of 10 mM N,N-diethyl-m-toluamide (DEET; HPLC external standard) andthe resulting solution analyzed by HPLC for MTSO (methyl-p-tolylsulfoxide), the stoichiometric oxidation product produced by reaction ofMTS with peracetic acid. A control reaction was run in the absence ofadded extract protein or triacetin to determine the rate of oxidation ofMTS in the assay mixture by hydrogen peroxide, for correction of therate of peracetic acid production for background MTS oxidation. HPLCmethod: Supelco Discovery C8 column (10-cm×4.0-mm, 5 μm) (catalog#569422-U) with Supelco Supelguard Discovery C8 precolumn(Sigma-Aldrich; catalog #59590-U); 10 microliter injection volume;gradient method with CH₃CN (Sigma-Aldrich; catalog #270717) anddeionized water at 1.0 mL/min and ambient temperature (Table 4).

TABLE 12 HPLC Gradient for analysis of peracetic acid. Time (min:sec) (%CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00 (stop) 40

For determination of the rate of perhydrolase-catalyzed acetic acidproduction in the reaction, a sample (0.900 mL) of the reaction mixturewas removed at a predetermined time and immediately added to a 1.5mL-microcentrifuge tube containing 0.040 mL of 0.75 M H₃PO₄, and theresulting solution briefly mixed to terminate the reaction at pH3.0-4.0. To the tube was then added 0.020 mL of a solution of 10 mg/mLof Aspergillus niger catalase (Sigma-Aldrich; C3515) in 50 mM phosphatebuffer pH (7.2), and the resulting solution mixed and allowed to reactfor 15 minutes at ambient temperature to disproportionate unreactedhydrogen peroxide to water and oxygen. To the tube was then added 0.040mL of 0.75 M H₃PO₄ and the resulting solution mixed and filtered usingan ULTRAFREE® MC-filter unit (30,000 Normal Molecular Weight Limit(NMWL), Millipore Corp., cat # UFC3LKT 00) by centrifugation for 2 minat 12,000 rpm. An aliquot (0.100 mL) of the resulting filtrate was mixedwith 0.150 mL of 20 mM MTS (methyl-p-tolyl sulfide) in acetonitrile, andthe resulting solution was incubated for 10 min at ca. 25° C. in theabsence of light. The concentration of acetic acid in the sampleproduced by both enzymatic hydrolysis of triacetin and conversion ofperacetic acid to acetic acid by reaction with MTS was determined usinga gas chromatograph (GC) equipped with a flame ionization detector (FID)and a DB-FFAP column (length, 15 m; ID, 0.530 mm; film thickness, 1.00μm); a fresh injection port liner was employed for each ratedetermination (total of eight sample analyses) to avoid build up ofphosphoric acid in the injection port liner over time.

The Thermotoga neapolitana acetyl xylan esterase variants had asignificantly-higher specific activity for perhydrolysis of triacetinthan the wild-type esterase (Table 13). The perhydrolysis/hydrolysisratios for the T. neapolitana acetyl xylan esterase variants weredetermined by dividing the rate of FAA production (perhydrolysis rate)by the rate of hydrolysis of triacetin to acetic acid (hydrolysis rate)(calculated from the rate of total acetic acid production in the assaymethod from both PM and acetic acid, and corrected for the rate ofperacetic acid production); the P/H ratio of the T. neapolitana acetylxylan esterase variants were ca. equal to or greater than the P/H ratiofor the T. neapolitana wild-type acetyl xylan esterase (Table 13).

TABLE 13 specific Thermotoga enzyme perhydrolysis hydrolysis activityneapolitana concen. rate rate P/H (U/mg perhydrolase (μg/mL) (mM/min)(mM/min) ratio protein) wild type 50 3.61 1.22 3.0 72 C277S 10 4.40 1.612.7 440 C277T 10 4.24 0.81 5.2 424 C277A 12.5 4.14 1.43 2.9 331 C277V12.5 3.70 0.88 4.2 296

Example 18 Expression of Thermotoga maritima Acetyl Xylan EsteraseVariants in E. coli KLP18

Plasmids comprising acetyl xylan esterase mutations as described inco-owned, co-filed, and copending U.S. patent application AttorneyDocket No. CL4392 US NA were prepared from wild type Thermotoga martima(SEQ ID NO: 16) by substituting at amino acid residue position 277 anAla, Val, Ser, or Thr (SEQ ID NO: 74). The plasmids were used totransform E. coli/KLP18 (Example 3). Transformants were grown in LBmedia at 37° C. with shaking up to OD_(600nm)=0.4-0.5, at which timeIPTG was added to a final concentration of 1 mM, and incubationcontinued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGEwas performed to confirm expression of the acetyl xylan esterase at20-40% of total soluble protein.

Example 19 Preparation of Cell Lysates Containing Semi-Purified T.Maritima Acetyl Xylan Esterase Variants

Cell cultures (prepared as described in Example 16) were grown using afermentation protocol similar to that described in Example 15 at a 1-Lscale (Applikon). Cells were harvested by centrifugation at 5,000×g for15 minutes then resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0supplemented with 1.0 mM DTT. Resuspended cells were passed through aFrench pressure cell twice to ensure>95% cell lysis. Lysed cells werecentrifuged for 30 minutes at 12,000×g, and the supernatant was heatedat 75° C. for 20 minutes, followed by quenching in an ice bath for 2minutes. Precipitated protein was removed by centrifugation for 10minutes at 11,000×g. SDS-PAGE indicated that the CE-7 enzyme comprisedapproximately 85-90% of the total protein in the preparation.

Example 20 Specific Activity and Perhydrolysis/Hydrolysis Ratio of T.maritima Acetyl Xylan Wild-Type Esterase and C277 Esterase Variants

Reactions (40 mL total volume) were run at 25° C. in phosphate buffer(50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100mM) and one of the following acetyl xylan esterase variants: T. maritimaC277S variant perhydrolase (0.010 mg/mL of heat-treated extract totalprotein from E. coli KLP18/pSW228/C277S), T. maritima C277T variantperhydrolase (0.010 mg/mL of heat-treated extract total protein from E.coli KLP18/pSW228/C277T), T. maritima C277A variant perhydrolase (0.0125mg/mL of heat-treated extract total protein from E.coli/KLP18/pSW228/C277A), and T. maritima C277V variant perhydrolase(0.0125 mg/mL of heat-treated extract total protein from E.coli/KLP18/pSW228/C277V) (prepared as described in Example 19).Reactions were stirred for only the first 30 seconds of reaction toinitially mix the reactants and enzyme.

A reaction was also run under identical conditions to that describedimmediately above using 0.050 mg/mL of heat-treated extract totalprotein isolated from E. coli KLP18/pSW228 (expressing Thermotogamaritima wild-type acetyl xylan esterase), where the heat-treatedextract supernatant was prepared according to the procedure of Example19.

Two samples from each of the reaction mixtures described above weresimultaneously withdrawn after the first minute of each reaction, andevery two minutes thereafter for fifteen minutes, where one of the twosamples was analyzed for peracetic acid using a modification of themethod described by Karst et al., supra, and the second sample wasanalyzed for total acetic acid produced from both enzymatic hydrolysisof triacetin and from subsequent conversion of peracetic acid in sampleto acetic acid by reaction with methyl-p-tolyl sulfide (MTS).

The Thermotoga maritima acetyl xylan esterase variants had asignificantly-higher specific activity for perhydrolysis of triacetinthan the wild type esterase (Table 14). The perhydrolysis/hydrolysisratios for the T. maritima acetyl xylan esterase variants weredetermined by dividing the rate of PAA production (perhydrolysis rate)by the rate of hydrolysis of triacetin to acetic acid (hydrolysis rate)(calculated from the rate of total acetic acid production in the assaymethod from both FAA and acetic acid, and corrected for the rate ofperacetic acid production); the P/H ratio of the T. maritima acetylxylan esterase variants were ca. equal to or greater than the P/H ratiofor the T. neapolitana wild-type acetyl xylan esterase (Table 14).

TABLE 14 specific Thermotoga enzyme perhydrolysis hydrolysis activitymaritima concen. rate rate P/H (U/mg perhydrolase (μg/mL) (mM/min)(mM/min) ratio protein) wild type 50 3.06 0.47 6.5 61 C277S 10 7.77 0.4816 777 C277T 10 6.93 1.05 6.6 693 C277A 10 4.27 0.088 48 427 C277V 104.25 0.062 68 425

Example 21 Peracetic Add Production Using Perhydrolases

Reactions (100 mL total volume) containing triacetin (2 mM), hydrogenperoxide (10 mM) and from 0.1 μg/mL to 2.0 μg/mL heat-treated cellextract protein (prepared as described above, where the heat-treatmentwas performed at 85° C. for 20 min) were run in 10 mM sodium bicarbonatebuffer (initial pH 8.1) at 20° C. Determination of the concentration ofperacetic acid in the reaction mixtures was performed according to themethod described by Karst at al., supra. The peracetic acidconcentrations produced in 1 min, 5 min, 20 min, 40 min and 60 min arelisted in Table 15.

TABLE 15 Dependence of peracetic acid (PAA) concentration onperhydrolase concentration in reactions containing triacetin (2 mM) andhydrogen peroxide (10 mM) in sodium bicarbonate buffer (10 mM, initialpH 8.1) at 20° C., using heat-treated extract protein from E. coliKLP18/pSW228 (Thermotoga maritima wild-type perhydrolase) or E. coliKLP18/pSW228/C277S (Thermotoga maritima C277S variant perhydrolase)(duplicate reactions). Thermotoga enzyme PAA, PAA, PAA, maritimatriacetin concen. 1 min 5 min 20 min PAA, 40 min PAA, 60 minperhydrolase (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm) no enzyme 2 0 00 1 1 3 wild type 2 0.2 0 2 7 13 19 wild type 2 0.2 0 1 5 11 15 wildtype 2 0.5 0 2 12 19 25 wild type 2 0.5 0 2 12 21 26 wild type 2 1.0 0 520 29 31 wild type 2 1.0 0 5 19 30 31 wild type 2 2.0 1 11 24 24 20 wildtype 2 2.0 1 11 29 29 21 C277S 2 0.2 0 4 18 18 18 C277S 2 0.2 0 4 18 1718 C277S 2 0.5 1 12 39 54 64 C277S 2 0.5 1 10 34 52 64 C277S 2 1.0 18 2659 69 63 C277S 2 1.0 18 25 60 70 64 C277S 2 2.0 9 38 66 60 48 C277S 22.0 9 34 69 61 49

Example 22 Peracetic Acid Production Using Perhydrolases

Reactions (100 mL total volume) containing triacetin (20 mM), hydrogenperoxide (10 mM) and from 0.1 μg/mL to 2.0 μg/mL heat-treated cellextract protein (prepared as described above, where the heat-treatmentwas performed at 85° C. for 20 min) were run in 10 mM sodium bicarbonatebuffer (initial pH 8.1) at 20° C. Determination of the concentration ofperacetic acid in the reaction mixtures was performed according to themethod described by Karst et al., supra. The peracetic acidconcentrations produced in 1 min, 5 min, 20 min, 40 min and 60 min arelisted in Table 16.

TABLE 16 Dependence of peracetic acid (PAA) concentration onperhydrolase concentration in reactions containing triacetin (20 mM) andhydrogen peroxide (10 mM) in sodium bicarbonate buffer (10 mM, initialpH 8.1) at 20° C., using heat-treated extract protein from E. coliKLP18/pSW228 (Thermotoga maritima wild-type perhydrolase) or E. coliKLP18/pSW228/C277S (Thermotoga maritima C277S variant perhydrolase)(duplicate reactions). Thermotoga enzyme PAA, PAA, PAA, maritimatriacetin concen. 1 min 5 min 20 min PAA, 40 min PAA, 60 minperhydrolase (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm) no enzyme 20 0 23 3 7 9 wild-type 20 0.2 3 10 15 27 35 wild-type 20 0.2 4 9 19 32 41wild-type 20 0.5 3 9 21 39 52 wild-type 20 0.5 3 8 22 39 54 wild-type 201.0 4 13 35 62 82 wild-type 20 1.0 4 12 37 67 wild-type 20 2.0 9 20 5291 122 wild-type 20 2.0 10 20 52 87 114 C277S 20 0.2 7 16 67 109 148C277S 20 0.2 9 24 67 112 144 C277S 20 0.5 16 43 140 202 260 C277S 20 0.517 48 148 228 272 C277S 20 1.0 24 75 230 289 353 C277S 20 1.0 26 97 232297 372 C277S 20 2.0 32 130 318 402 443 C277S 20 2.0 37 135 323 401 430

Example 23 Peracetic Acid Production Using Perhydrolases

Reactions (40 mL total volume) were run at 25° C. in phosphate buffer(50 mM, pH 7.2) containing triacetin (100 mM), hydrogen peroxide (100mM) and from 10 μg/mL to 50 μg/mL of heat-treated T. neapolitana or T.maritima wild-type or C277 variant perhydrolases (as heat-treated cellextract protein prepared as described above, where the heat-treatmentwas performed at 75° C. for 20 min). Reactions were stirred for only thefirst 30 seconds of reaction to initially mix the reactants and enzyme.Determination of the concentration of peracetic acid in the reactionmixtures was performed according to the method described by Karst etal., supra. The peracetic acid concentrations produced in 1 min, 5 min,and 30 min are listed in Table 17.

TABLE 17 Peracetic acid (PAA) production in reactions containingtriacetin 100 mM) and hydrogen peroxide (100 mM) in phosphate buffer (50mM, pH 7.2) at 25° C., using heat-treated T. neapolitana or T. maritimawild-type or C277 variant perhydrolases. enzyme PAA, PAA, PAA, triacetinH₂O₂ concen. 1 min 5 min 30 min perhydrolase (mM) (mM) (μg/mL) (ppm)(ppm) (ppm) no enzyme 100 100 0 63 54 80 T. maritima 100 100 50 529 17903785 wild-type T. maritima 100 100 10 979 3241 4635 C277S T. maritima100 100 10 933 2882 3527 C277T T. maritima 100 100 10 442 2018 2485C277A T. maritima 100 100 10 577 1931 2278 C277V T. neapolitana 100 10050 514 1837 3850 wild-type T. neapolitana 100 100 10 606 2237 4609 C277ST. neapolitana 100 100 10 634 2198 3918 C277T T. neapolitana 100 10012.5 516 2041 3735 C277A T. neapolitana 100 100 12.5 451 1813 2758 C277V

Example 24 Peracetic Add Production Using Perhydrolases

Reactions (10 mL total volume) were run at 25° C. in sodium bicarbonatebuffer (1 mM, initial pH 6.0) containing triacetin (100 mM or 150 mM),hydrogen peroxide (100 mM, 250 mM or 420 mM) and heat-treated T.neapolitana or T. maritima wild-type, C277S or C277T variantperhydrolases. Reactions run using 420 mM hydrogen peroxide additionallycontained 500 ppm TRUPINAL® SL. Reactions were stirred for only thefirst 30 seconds of reaction to initially mix the reactants and enzyme.Determination of the concentration of peracetic acid in the reactionmixtures was performed according to the method described by Karst etal., supra. The peracetic acid concentrations produced in 1 min, 5 min,and 30 min are listed in Table 18.

TABLE 18 Peracetic acid (PAA) production in reactions containingtriacetin and hydrogen peroxide in bicarbonate buffer (1 mM at pH 6.0 or100 mM at pH 8.1) or in deionized water (pH 5.0) at 25° C. usingheat-treated T. maritima wild- type, C277S or C277T variantperhydrolases. Thermotoga NaHCO₃ PAA, PAA, PAA, maritima triacetin H₂O₂buffer enzyme concen. 1 min 5 min 30 min perhydrolase (mM) (mM) (mM)(μg/mL) (ppm) (ppm) (ppm) no enzyme 100 100 1.0 0 28 78 141 wild-type100 100 1.0 75 434 494 608 wild-type 100 100 1.0 100 449 667 643 C277S100 100 1.0 15 989 1554 1476 C277S 100 100 1.0 20 1301 2139 2131 C277T100 100 1.0 15 1062 1513 1393 C277T 100 100 1.0 20 996 1430 1516 noenzyme 100 250 0 0 13 71 71 wild-type 100 250 0 75 512 535 533 wild-type100 250 0 100 576 668 654 C277S 100 250 0 15 653 671 675 C277S 100 250 020 943 927 903 C277T 100 250 0 15 717 711 765 C277T 100 250 0 20 730 755743 no enzyme 150 420 100 0 417 810 848 wild-type 150 420 100 500 63038627 9237 C277S 150 420 100 100 7822 10349 10197

1. A multi-component system for producing a peroxycarboxylic acidcomprising (a) providing a first component comprising: (i) a carboxylicacid ester substrate; (ii) an enzyme catalyst having perhydrolysisactivity, wherein said enzyme catalyst comprises an enzyme having a CE-7signature motif that aligns with SEQ ID NO: 2 using CLUSTALW, saidsignature motif comprising: (1) an RGQ motif at amino acid positionsaligning with 118-120 of SEQ ID NO:2; (2) a GXSQG motif at amino acidpositions aligning with 179-183 of SEQ ID NO:2; and (3) an HE motif atamino acid positions aligning with 298-299 of SEQ ID NO:2; said enzymecomprising at least 90% amino acid identity to SEQ ID NO: 2, 14, 16, 73or 74; and (iii) at least one cosolvent comprising an organic solventhaving a log P of less than about 2, wherein log P is defined as thelogarithm of the partition coefficient of a substance between octanoland water, expressed as P=[solute]_(octanol)/[solute]_(water) andwherein the cosolvent is not a substrate for said enzyme catalyst;wherein said first component is a substantially non-aqueous formulationof (i)-(iii); and (b) providing a second component comprising a sourceof peroxygen in water; wherein said first component and said secondcomponent are combined to produce an aqueous reaction formulationcomprising a peroxycarboxylic acid and wherein said cosolventsolubilizes the carboxylic acid ester substrate in the aqueous reactionformulation without substantial loss of perhydrolytic activity of theenzyme catalyst.
 2. The system according to claim 1 further comprising afirst compartment for storing said first component and a secondcompartment for storing said second component.
 3. The system accordingto claim 2 further comprising a mixing compartment for receiving atleast some of said first component from said first compartment and atleast some of said second component from said second compartment,thereby permitting the formation of a mixture comprising at least someof said first component and at least some of said second component. 4.The system according to claim 3 wherein the at least some of said firstcomponent is mixed with the at least some of said second component in aratio of about 1:1 to about 1:10 by weight.
 5. The system according toclaim 4 further comprising a nozzle for dispensing the aqueous reactionformulation from said mixing compartment.
 6. The system according toclaim 5 further comprising a nozzle for receiving at least some of saidfirst component from said first compartment and at least some of saidsecond component from said second compartment, and for dispensing atleast some of said first component contemporaneously with at least someof said second component.
 7. The system according to claim 1 wherein thefirst component and the second component are delivered to a surface witha nozzle permitting the mixing of the first component and the secondcomponent on the surface.